WO1999054445A2 - Traitement associe utilisant des acides nucleiques et des medicaments classiques - Google Patents

Traitement associe utilisant des acides nucleiques et des medicaments classiques Download PDF

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Publication number
WO1999054445A2
WO1999054445A2 PCT/CA1999/000371 CA9900371W WO9954445A2 WO 1999054445 A2 WO1999054445 A2 WO 1999054445A2 CA 9900371 W CA9900371 W CA 9900371W WO 9954445 A2 WO9954445 A2 WO 9954445A2
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Prior art keywords
cell cycle
cells
nucleic acid
gene
cell
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PCT/CA1999/000371
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English (en)
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WO1999054445A3 (fr
Inventor
Phalgun B. Joshi
Ian C. Mortimer
Patrick M. S. Tam
Ian Maclachlan
Roger W. Graham
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Inex Pharmaceuticals Corporation
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Priority claimed from US09/295,663 external-priority patent/US6841537B1/en
Application filed by Inex Pharmaceuticals Corporation filed Critical Inex Pharmaceuticals Corporation
Priority to EP99917711A priority Critical patent/EP1082419A2/fr
Priority to JP2000544777A priority patent/JP2002512258A/ja
Priority to AU35912/99A priority patent/AU762986B2/en
Priority to CA002325561A priority patent/CA2325561A1/fr
Publication of WO1999054445A2 publication Critical patent/WO1999054445A2/fr
Publication of WO1999054445A3 publication Critical patent/WO1999054445A3/fr

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0038—Radiosensitizing, i.e. administration of pharmaceutical agents that enhance the effect of radiotherapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Definitions

  • the present invention relates to methods of enhancing the therapeutic effect of a therapeutic nucleic acid by combining treatment with a conventional drug that is a cell cycle blocking agent.
  • the present invention also pertains to methods for increasing the efficiency of transformation of cycling cells.
  • the invention further relates to cancer therapy, and, in particular, to methods of efficiently transforming cancer cells with nucleic acids that encode gene products that inhibit the growth of cancer cells.
  • the cell cycle consists of a cell division phase and the events that occur during the period between successive cell divisions, known as interphase. Most cell components are made continuously throughout interphase. As such; it is difficult to define distinct stages in the progression of the growing cell through interphase.
  • S synthesis
  • the other distinct stage of the cell cycle is the cell division phase, which includes both nuclear division (mitosis) and the cytoplasmic division (cytokinesis) that follows.
  • G gap
  • G 2 phase The period between the completion of DNA synthesis and the next M phase.
  • Nuclear membrane dissolution generally takes place immediately prior to cell division, during the M phase or the G 2 /M interphase.
  • interphase is composed of successive Gi, S, and G 2 phases. The duration of an entire cycle varies from cell type to cell type, but is often about 24 hours.
  • the cell contains extremelyly sensitive feedback control circuits that regulate entry into, exit out of and the events that occur during a given phase of the cell cycle. These circuits can, for example, prevent exit from the S phase if a fraction of a percent of DNA remains unreplicated, and they can block advance into anaphase in mitosis until all the chromosomes have aligned on the metaphase plate.
  • the progression of a cell through the mitotic cycle is controlled by an array of regulatory factors that act as "checkpoints" and assure that the previous stage has been completed before the subsequent stage ensues.
  • the relative abundance of these factors oscillates as the cell cycle advances, either by synthesis of a particular gene product or by chemical transformation, such as phosphorylation and dephosphorylation events.
  • cyclin-dependent kinases a family of master enzymes, i.e., cyclin-dependent kinases (see, Sherr, Cell, 73:1059-1065 (1993)). These enzymes are composed of two proteins, a regulatory subunit (the cyclin) and an associated catalytic subunit (the actual cyclin-dependent kinase or CDK), the levels of which vary with different phases of the cell cycle (see, Peters, G., Nature, 371:204-205 (1994)). Both cyclins and CDKs represent molecular families that encompass a variety of genetically related, but functionally distinct proteins.
  • cycling cells can be synchronized at a specific stage of the cell cycle by growth factor deprivation(see, Keyomarsi, et al., Cancer Res,. 51:3602-3609 (1991).
  • PCT Publication No. WO 94/00095 discloses the use of various calpain inhibitors to synchronize the cell cycle. Synchronization of cells, by itself, is a research tool that is not known to be useful for treating disease in a patient. It is known, however, that compounds that induce cell cycle arrest (e.g. , hydroxyurea, VM-26, cisplatin and taxol) can be used therapeutically for cancer treatment.
  • compounds that induce cell cycle arrest e.g. , hydroxyurea, VM-26, cisplatin and taxol
  • cancer cells are among the most rapidly proliferating cells in multicellular organisms, and that they would therefore be more susceptible to compounds that disrupt or arrest cell cycling.
  • conventional drugs including cell cycle blocking agents, for the treatment of cancer.
  • Various combinations of conventional drugs known in the art are as follows: for acute lymphocytic leukemia - vincristine, prednisone, doxorubicin and L-asparaginase; for Hodgkin's disease - mechoroethamine, vincristine, procarbazine and prednisone (MOPP); for histiocytic lymphoma - cyclophosphamide, vincristine, procarbazine and prednisone (C-MOPP); and for testicular carcinoma - bleomycin, vinblastine, and cisplatin.
  • SPLPs are fully encapsulated lipid-plasmid particles that are resistant to nuclease degradation, have low immunogenicity, and are of small size ( ⁇ 150 nm), thereby making them particularly well suited for long circulation lifetimes.
  • Therapeutic uses for these SPLPs have been disclosed in U.S. Patent Applications Serial Nos. 60/063,473; 60/073,598; 60/082,665; 60/086,917, all of which are assigned to the assignee of the instant invention and are incorporated herein by reference.
  • Son and Huang Proc. Natl. Acad. Sci, 91 :12669-12672 (1994), reported that improved expression of a plasmid delivered directly to tumor cells can be obtained in tumor cells seven days after treatment with cisplatin. Other drugs, including vincristine, were not found to be effective. Son and Huang did not teach, suggest or appreciate the benefits associated with the synchronization of cells at the treatment site for enhanced use of therapeutic gene drugs. Further, Son and Huang neither taught nor appreciated that the synchronization of cells could be used to increase the efficiency of transformation, nor did they teach or suggest that synchronized cells at certain stages of the cell cycle are more efficiently transformed with therapeutic genes.
  • the present invention provides methods of increasing the efficiency of cellular transformation of cycling cells, the methods comprising synchronizing cycling cells at a first stage of the cell cycle, and transforming these cells at a second stage within about an additional cell cycle with a genetically engineered nucleic acid that encodes a desired gene product.
  • the invention provides methods for killing a cycling cell, the methods comprising contacting a target population of cells with a first agent that synchronizes (for example, by transiently blocking progression of the cell cycle) the cells, and thereafter contacting the cell with a nucleic acid that transforms the cell within about an additional cell cycle.
  • the first agent blocks the progression of the Gi, S, G 2 , or mitosis stage of the cell cycle.
  • the nucleic acid is preferably part of a lipid-nucleic acid particle (lipoplex) and encodes a desired gene product.
  • the duration of contact with the first agent is advantageously limited to a first time period sufficient to block the progression of the cell cycle at a particular stage of the cell cycle or to synchronize cells at a particular stage of the cell cycle.
  • the duration of contact with the nucleic acid is advantageously limited to a second time period whose timing and duration are sufficient for cells to be transformed.
  • improved transfection of cells at the site of neoplasm by therapeutic genes is achieved after a period of cell cycle blocking, particularly at the G 2 /M interphase or the M phase.
  • cell cycle blocking particularly at the G 2 /M interphase or the M phase.
  • maximal luciferase transgene expression was found to coincide with the transition of cells from G 2 /M phase into the Gi phase of the subsequent cell cycle.
  • cells are most amenable to transfection when the cells are incubated with lipoplexes during or just before mitosis.
  • Plasmids may be more successfully delivered to the nucleus (the site of transcription) during this period, perhaps because of the limited amount of time plasmids are exposed to intra-cellular nucleases and other degradation elements before they are localized in the nucleus and transcribed. Alternatively, though less likely, the cells may be taking up, such as by endocytosis, more of the delivered plasmid during the period of cell cycle blockage, thus resulting in a proportionately higher expression.
  • transfection preferably occurs in cells in the G 2 M phase. Such cells may represent less than 10% of the population at any given time. The remaining 90% either resist uptake of plasmid, or degrade or inhibit the plasmid before it is expressed in the nucleus.
  • a greater population of cells are synchronized in a transfection/expression competent state at the time of exposure to the plasmid thereby resulting in improved expression of the gene at the tumor site.
  • the methods of the present invention are not limited to the transfection of malignant cells at the site of neoplasm, but can also include benign cells at the site of neoplasm.
  • benign cells include macrophages and immune system cells, as well as normal healthy vascular endothelial cells which are induced by tumorigenic ones.
  • the choice of therapeutic gene may be selected according to the cell that is actually transfected. For example, if the vascular endothelium is targeted, then a therapeutic gene such as the gene encoding endostatin or angiostatin will successfully inhibit tumor growth.
  • the present invention is particularly useful when the anti-neoplastic drug is combined with the delivery of the herpes simplex virus thymidine kinase gene (HSV- TK), followed by treatment with the pro-drug ganciclovir.
  • HSV-TK converts the pro- drug into a toxic analog (described in detail in U.S. Patent Application Serial No. 60/073,598, the teachings of which are incorporated herein by reference). Transfection with HSV-TK results in a "bystander effect," wherein either the TK gene product or the toxic analog diffuses into neighboring cells where the toxic effect is also exerted. This system allows for greater cell killing from a limited number of transfection sites.
  • the present invention also encompasses improved methods for transfection of cells at organs or disease sites which are not cancerous. Using these methods, alternative therapeutic genes may be employed at these sites. For example, improved transfection of spleen cells using a gene encoding an immune stimulating peptide, such as IL-12, is taught herein.
  • an immune stimulating peptide such as IL-12
  • the methods of the invention have self-evident utility as methods of improving tumor response to gene therapy and, more broadly, as methods of treating cancer in a patient.
  • Figure 2 DNA uptake in SK-OV-3 cells. Shown are histograms of relative YOYO-1 iodide/DNA fluorescence versus number of cell obtained by flow cytometry. Plasmid DNA was labeled with YOYO-1 iodide and formulated as lipoplexes. After a 16 hr incubation with aphidicolin to arrest cells in the Gi phase of the cell cycle, cultures were incubated with DODAC:DOPE:plasmid lipoplexes for 1 hr in the continuous presence of aphidicolin (+/+) or in the absence of aphidicolin to remove the cell cycle arrest (+/-).
  • FIG. 3 Effect of aphidicolin on gene expression in HeLa-luc cells.
  • HeLa-luc cells were arrested in the Gi phase of the cell cycle by the administration of aphidicolin as described in the Example Section. Luciferase gene expression was measured at 0, 6 and 24 hours after arrest for both untreated cells (open bars) and cells blocked in the Gj (solid bars) phase of the cell cycle.
  • Figure 4 Effect of cell cycle arrest on lipoplex mediated transfection.
  • Cells were arrested in the Gj phase of the cell cycle by treatment with aphidicolin, incubated with 0.5 ⁇ g plasmid containing lipoplexes that encoded luciferase, and assayed for luciferase activity.
  • (-/-) represents control cultures that have never been exposed to aphidicolin; (+/-) represents cultures that were synchronized with treatment with aphidicolin, but then incubated with lipoplexes in the absence of aphidicolin; (+/+) represents cultures that were synchronized with treatment with aphidicolin and were then incubated with lipoplexes in the continued presence of aphidicolin.
  • Luciferase activity was measured after 24 hours of incubation with lipoplexes. The data is presented as the mean of triplicate samples ⁇ s.d.
  • FIG. 5 Correlation of kinetics of luciferase gene expression in aphidicolin treated cells allowed to progress through the cell cycle.
  • SK-OV-3 cells were arrested as described in the Example Section.
  • Asynchronous cultures that had never been exposed to aphidicolin (-/-) ( ⁇ ) were used as controls.
  • Samples were harvested at 2 hr intervals and analyzed for luciferase activity as well as cell cycle status (C). Analysis of cell cycle status was determined at the time points shown as described in the Example Section. Luciferase expression is expressed as the mean of triplicate samples ⁇ s.d.
  • SK-OV-3 cells were arrested as described in the Example Section. At time 0, arrested cells were released from the cell cycle block by the addition of fresh media without aphidicolin; blocked cells were supplemented with aphidicolin in fresh growth media.
  • Luciferase was measured every two hours for untreated cells ( ⁇ ), cells released from the aphidicolin block ( ⁇ ) and cells that were arrested throughout the experiment (A). Luciferase expression is expressed as the mean of triplicate samples.
  • Figure 8(A) illustrates the pINEX L018 plasmid construct description and map
  • Figure 8(B) illustrates the pINEX TK10 plasmid construct
  • Figure 9 Cell cycle status of cells at tumor site (MCA 207) in response to treatment with vincristine.
  • MCA 207 normal chromosome complement.
  • Empty SM/Chol liposomes (control) Figure 9(A)
  • 24h after treatment with OncoTCS Figure 9(B)
  • 48 h after treatment with OncoTCS i.e., liposome + vincristine
  • Figure 10 Effect of Pretreatment of OncoTCS (i.v. 0.5 mg/kg) on transfection of MCA 207 tumors using INEX TCS 351 (It. 2 ⁇ g) (Tumors harvested after 12 h).
  • Figure 11 Effect of OncoTCS pretreatment (i.v., 0.5 mg/kg) on transfection after it. administration of INEX 351 (2 ⁇ g DNA) into U87 subcutaneous tumors.
  • Figure 12 Effect of length of interval between OncoTCS (i.v. 0.5 mg/kg) treatment and INEX 324 (i.v., 75 ⁇ g DNA) administration in spleen cells harvested 8 h after plasmid administration.
  • Figure 13 Effect of X-rays on luciferase activity in i.p. B16 tumors after i.p. administration of INEX 324.
  • Figure 14 sets forth the schedule of vincristine administrations.
  • Figure 15 illustrates the effect of vincristine on transfection using INEX 303 on neuro 2a tumors.
  • Figure 16 illustrates luciferase expression at the in vivo tumor site 48 h after INEX 303 administration and 0, 32 h and 16 h after OncoTCS administration.
  • the present invention relates to methods for enhancing the therapeutic effects of a therapeutic nucleic acid by combining treatment with a conventional drug , X- rays or other form of radiation, all of which are cell cycle blocking agents.
  • the invention takes advantage of the surprising discovery that more efficient transformation of cells by therapeutic nucleic acids delivered to the cells (/. e. , in a plasmid construct) can be achieved by minimizing the time between transfection of the plasmid and expression of plasmid in the nucleus.
  • the methods of the invention are achieved by delivering the plasmid to cells that are synchronized at a stage in the cell cycle when the nuclear membrane is substantially dissolved, generally just prior to cell division.
  • the cells at the disease or target site of interest can be synchronized by pretreatment with a drug or with some form of radiation (such as X-ray) that is a cell cycle blocking agent.
  • therapeutic nucleic acids which are delivered according to the invention are rapidly expressed in the cell nucleus and spend a reduced amount of time exposed to intracellular nucleases and other degradative metabolic processes; thereby resulting in a more efficient transformation of these cycling cells and an enhanced therapeutic effect of the therapeutic nucleic acid.
  • DC-Choi 3 ⁇ -(N-(N',N'- dimethylaminoethane)carbamoyl)cholesterol (see, Gaoet, et al, Biochem. Biophys. Res.
  • DDAB N,N-distearyl-N.N-dimethylammonium bromide
  • DMRIE N-(l ,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxy ethyl ammonium bromide
  • DODAC N,N-dioleyl-N,N-dimethylammonium chloride
  • DOGS diheptadecylamidoglycyl spermidine
  • DOPE 1,2- stt-dioleoylphoshatidyethanolamine
  • DOSPA N-(l -(2,3-dioleyloxy)propyl)-N-(2- (speirninecarboxaii_ido)e1_hyl)-N,N-dimethylammonium trifluoroacetate
  • DOTAP N-(l- (2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride
  • DOTMA N-(l -(2,3- dioleyloxy)propyl)-N,N,N-trimethylammonium chloride
  • EPC egg phosphatidylcholine
  • RT room temperature
  • HEPES 4-(2-hydroxyethyl)-l-pipe
  • POPC palmitoyl oleoyl phosphatidylcholine (Northern Lipids, Vancouver, BC); QELS, quasielastic light scattering; TBE, 89 mM Tris-borate with 2 mM EDTA; and EDTA, Ethylenediaminetetraacetic acid (Fisher Scientific, Fair Lawn, NJ).
  • acyl refers to a radical produced from an organic acid by removal of the hydroxyl group.
  • acyl radicals include, but are not limited to, acetyl, pentanoyl, palmitoyl, stearoyl, myristoyl, caproyl and oleoyl.
  • lipid refers to any fatty acid derivative that is capable of forming a bilayer such that a hydrophobic portion of the lipid material orients toward the bilayer while a hydrophilic portion orients toward the aqueous phase.
  • Hydrophilic characteristics can be derived from the presence of phosphato, carboxylic, sulfato, amino, sulfhydryl, nitro, and other like groups. Hydrophobicity can be conferred by the inclusion of groups that include, but are not limited to, long chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic or heterocyclic group(s).
  • Preferred lipids are phosphoglycerides and sphingolipids, representative examples of which include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine or dilinoleoylphosphatidylcholine could be used.
  • lipid Other compounds lacking in phosphorus, such as sphingolipid and glycosphingolipid families are also within the group designated as lipid. Additionally, the amphipathic lipids described above may be mixed with other lipids including triglycerides and sterols.
  • non-cationic lipid refers to any of a number of lipid species which exist either in an uncharged form, a neutral zwitterionic form or an anionic form at physiological pH.
  • lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cardiolipin and cerebrosides.
  • cationic lipid refers to any of a number of lipid species that carry a net positive charge at physiological pH. Such lipids include, but are not limited to,
  • DODAC DODAC
  • DOTMA DOTMA
  • DDAB DDAB
  • DOTAP DC-Choi
  • DMRIE DC-Choi
  • cationic lipids are available which can be used in the present invention. These include, for example, LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and DOPE, from GIBCO/BRL, Grand Island, New York, USA); LIPOFECTAMINE® (commercially available cationic liposomes comprising
  • DOSPA and DOPE from GIBCO/BRL
  • TRANSFECTAM® commercially available cationic liposomes comprising DOGS from Promega Corp., Madison, Wisconsin, USA.
  • cellular transformation or, interchangeably, “transfection,” as used herein, refers to the introduction of polyanionic materials, particularly nucleic acids, into cells.
  • lipofection refers to the introduction of such materials using liposome or lipid-based complexes.
  • the polyanionic materials can be in the form of DNA or RNA that is linked to expression vectors to facilitate gene expression after entry into the cell.
  • the polyanionic material used in the present invention is meant to include DNA having coding sequences for structural proteins, receptors and hormones, as well as transcriptional and translational regulatory elements (i. e. , promoters, enhancers, terminators and signal sequences) and vectors.
  • transcriptional and translational regulatory elements i. e. , promoters, enhancers, terminators and signal sequences
  • vectors Methods of incorporating particular nucleic acids into expression vectors are well known to those of skill in the art, but are described in detail in, for example, Sambrook, et al, MOLECULAR CLONING: A LABORATORY MANUAL (2nd ed.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989) or CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, F. Ausubel, et al., ed. Greene Publishing and Wiley-Interscience, New York (1987), both of which are incorporated herein by reference.
  • Coding vectors or, interchangeably, “vectors,” as used herein, refer to viruses, plasmids or other nucleic acid molecules that are able to replicate in a chosen host cell.
  • Expression vectors may replicate autonomously, or they may replicate by being inserted into the genome of the host cell using methods well known in the art.
  • Vectors that replicate autonomously will have an origin of replication or autonomous replicating sequence (ARS) that is functional in the chosen host cell(s).
  • ARS autonomous replicating sequence
  • “Expression vectors” contain all of the control elements for expression of gene products encoded by the vectors.
  • Conditions for "increasing the efficiency of cellular transformation” refer to conditions under which a higher percentage of cells in a target cell population (for example, the cells of a tumor) are transformed relative to a control population of cells. Preferably, at least 50 % more cells, more preferably at least 2 times more cells and, even more preferably, greater than 10 times more cells are transformed relative to the control cells.
  • a stage of the cell cycle when the nuclear membrane is substantially degraded is generally late G 2 to M phase, but may be earlier. This may be ascertained by microscopic examination.
  • Cycling cells are cells that are not permanently arrested in any portion of the cell cycle.
  • Synchronizing cells at a stage of the cell cycle means that a population of cells that are at different stages of the cell cycle are induced to remain at, or proceed to, a particular phase of the cell cycle, such that at least 10%, preferably 30%, more preferably 60%, and most preferably more than 90% of the cells are all in the same stage of the cell cycle. Synchronizing generally means increasing the fraction of cells that are at a certain stage of the cell cycle. In a preferred embodiment, compounds or conditions that synchronize cells do so by arresting cells in a particular stage of the cell cycle.
  • a “cell cycle blocker” or, interchangeably, a “cell cycle blocking agent” is a synchronizing compound or a radiation treatment, such as X-rays, that inhibits a cell from proceeding into a subsequent cell cycle phase to which the cell would proceed in the absence of the compound.
  • cell cycle blockers arrest the cell cycle without adversely affecting cellular processes (such as endocytosis) related to transformation, such as the uptake of DNA and gene expression.
  • nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer, et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka, et al., J. Biol. Chem. 260:2605-2608 (1985); and Cassol, et al., 1992; Rossolini, et al, Mol. Cell. Probes 8:91-98 (1994)).
  • the term nucleic acid is used interchangeably with gene, cDNA and mRNA encoded by a gene. Unless otherwise indicated, a particular nucleic acid sequence includes the perfect complementary sequence thereof.
  • nucleic acids used in the method of the present invention can be isolated from natural sources, obtained from such sources as ATCC or GenBank libraries or prepared by synthetic methods. Synthetic nucleic acids can be prepared by a variety of solution or solid phase methods. Generally, solid phase synthesis is preferred. Detailed descriptions of the procedures for solid phase synthesis of nucleic acids by phosphite-triester, phosphotriester, and H-phosphonate chemistries are widely available. See, for example, Itakura, U.S. Patent No. 4,401,796; Caruthers, et !., U.S. Patent Nos.
  • Nucleic acid probes or “primers” can be DNA or RNA fragments.
  • DNA fragments can be prepared, for example, by digesting plasmid DNA, or by use of PCR, or synthesized by either the phosphoramidite method described by Beaucage and Carruthers, Tetrahedron Lett., 22:1859-1862 (1981), or by the triester method according to Matteucci, et al., J. Am. Chem. Soc, 103:3185 (1981), both of which are incorporated herein by reference.
  • a double stranded fragment may then be obtained, if desired, by annealing the chemically synthesized single strands together under appropriate conditions or by synthesizing the complementary strand using DNA polymerase with an appropriate primer sequence.
  • a specific sequence for a nucleic acid probe is given, it is understood that the complementary strand is also identified and included. The complementary strand will work equally well in situations where the target is a double- stranded nucleic acid.
  • a nucleic acid sequence encoding refers to a nucleic acid that contains sequence information for a structural RNA, such as a rRNA, a tRNA, or the primary amino acid sequence of a specific protein or peptide, or a binding site for a transacting regulatory agent. This phrase specifically encompasses degenerate codons (i.e., different codons which encode a single amino acid) of the native sequence or sequences which may be introduced to conform with codon preference in a specific host cell.
  • the “gene product of the nucleic acid” refers to an mRNA, structural RNA such as rRNA, a tRNA, or a specific protein or peptide. This gene product may be directly or indirectly toxic. It may induce apoptosis or differentiation, or the transcript alone may incapacitate the target cell (i.e., a ribozyme or antisense product; or a protein with "nuclear jamming" ability).
  • the term "effective amount” means an amount or dosage of a given substance that is sufficient to produce a desired result.
  • the desired result can be a subjective or objective improvement, such as a clinical improvement, in the recipient of the dosage, a decrease in tumor size, a decrease in the rate of growth of cancer cells, or a decrease in metastasis.
  • Toxic means that a compound has a deleterious effect on target cells, such as cancer cells. A toxic compound kills, slows the growth and/or alters the metabolism of the target cell.
  • a "therapeutic gene” is one whose gene product performs a clinically useful function. For example, where the therapeutic gene is used to transform cancer cells, the therapeutic gene will inhibit the growth of the cancer cells.
  • the therapeutic gene is preferably one whose gene product has low toxicity to non-target tissues, and high toxicity to the disease (e.g. cancer) site.
  • the gene product when delivered in the preferred lipid-nucleic acid (e.g., lipid-plasmid particles) particles of the invention, the gene product preferably has greater toxicity to tumor cells than liver or spleen cells, where a large portion of particles can normally be cleared.
  • a therapeutic gene may be delivered to a treatment site, which is not a disease site, but which activates an immunologic or other response which is then favorable for the amelioration of the disease or disorder being treated.
  • therapeutic genes useful in the methods of the present invention include, but are not limited to, genes for: pro-apoptotic proteins; tumor suppressors (e.g., p53, Rbl (Retinoblastoma), etc.); cytokines (such as Interleukin-2, Interleukin-12, etc.); heat shock proteins; immunogenic antigens (such as tumor-specific proteins, etc.); genes activated in embryos only; Endostatin, Angiostatin, Thrombospondin, and other inhibitors of angiogenesis;
  • tumor suppressors e.g., p53, Rbl (Retinoblastoma), etc.
  • cytokines such as Interleukin-2, Interleukin-12, etc.
  • heat shock proteins immunogenic antigens (such as tumor-specific
  • Enzymes used in GDEPT combinations i.e., suicide genes used in conjunction with a non- toxic pro-drug
  • a non- toxic pro-drug such as Thymidine Kinase from Herpes simplex virus (HSV-TK); cytosine deaminase; porfirin; TIMP-2 (tissue inhibitor of metallo proteinase-2); plant, bacterial or fungal toxin genes, such as saporin, ricin, diphtheria toxin, cholera toxin; viral protein genes, such as El A; mutated E6; SV40 Tag or viral protein genes which effect plasmid maintenance and/or copy number, such as EBNA-1 ; transcription plasmids encoding ribozymes or antisense oligonucleotides, Adenosine Deaminase; CFTR - Cystic Fibrosis; GM-CSF, IL-4.
  • HSV-TK Herpes simplex virus
  • IL-2, IL-7, IL-10 Carcineombryonic Antigen; HLA-B7; TNF; T-Cell Receptor Antibody; CEA; Ig; IFN-g; MART-1; Chimeric Antibody /TCR; Prostate Specific Antigen; anti-erbB-2; Single Chain Antibody; BRCA-1 ; Alpha-1 Antitrypsin; p47 phax; Fanconi Anemia Complementation Group C; Glucocerbrosidase; Iduronato-2- Sulfatase; Purine Nulceaside Phosphorylase.
  • Other therapeutic genes are continually being discovered and can be used in the methods of the present invention. Therapeutic genes are generally delivered as part of an expression construct, although other formats are possible.
  • a “foreign therapeutic gene” is a therapeutic gene that is introduced into a cell using genetic engineering techniques.
  • the term “foreign gene” can include genetically engineered additional copies of genes where the transformed cell has an endogenous copy of the gene.
  • a foreign gene can be a gene that is genetically engineered to be operably linked to a different promoter than the promoter of the endogenous gene.
  • Heterologous genes may be advantageous because their gene products may also serve to induce an immune response. For example, genes used in a suicide gene/prodrug system may have this effect.
  • Apoptosis is a process of programmed cell death that is defined by a number of characteristic phenomena, summarized in Cohen, Immunol. Today 14:126-30 (1993). For example, in apoptotic cells, the cytoplasm condenses, and the endoplasmic reticulum dilates to form vesicles which fuse with the cell membrane, producing characteristic cellular morphology. Changes in the nuclei include nuclear condensation, the formation of dense crescent-shaped aggregates of chromatin, nucleolus fragmentation, and formation of vesicles at or on the nuclear membrane. A classic signature of apoptosis is the cleavage of nuclear DNA into nucleosomal subunits.
  • endonucleases present in the cell cut the DNA in the linker regions between nucleosomes to release DNA fragments in integer multiples of 180-190 base pairs.
  • the pattern of cleavage is believed to result from the vulnerability of the linker DNA between the nucleosomes to endonucleases. On gels, this gives rise to the appearance of a ladder as nucleosomal units are sequentially cleaved from the DNA. Observation of a classic DNA ladder is indicative of apoptosis. For example, cells are lysed and the high molecular DNA is removed by centrifugation. The aqueous phase is treated with proteinase K to digest proteins.
  • the DNA is precipitated with salt and ethanol.
  • the pellet is dissolved in deionized water and treated with about 500 ⁇ g/mL RNase A.
  • the DNA is run on a 2% agarose minigel. Observation for a classic DNA ladders is made. A gel photograph can be taken. Cell death is verified by the demonstration of DNA fragmentation as represented by the ladder configurations on the gel (see, Gavrieli, Y., et al., J. Cell Biol. 119:493 (1992)).
  • There are also a variety of other assays available for apoptosis such as "TUNEL” assays (see, White, et al., J. Virol. 52:410 (1984)).
  • Growth inhibition may be assessed using a number of commonly used assays, such as the methylcellulose assay (see, e.g., Lunardi- Iskandar, et al., Clin. Exp. Immunol. 60:285-293 (1985)).
  • methylcellulose assay see, e.g., Lunardi- Iskandar, et al., Clin. Exp. Immunol. 60:285-293 (1985)).
  • tumor cell or "cancer cell” or “neoplastic cell” denotes a cell that demonstrates inappropriate, unregulated proliferation.
  • a cell line is said to be “malignant” if, when the cell line is injected into a host animal, the host animal develops tumors or cancers that are anaplastic, invasive and/or metastatic.
  • a "human” tumor is comprised of cells that have human chromosomes. Such tumors include those in a human patient, and tumors resulting from the introduction of a human malignant cell line into a nonhuman host animal if cells from such tumors have human chromosomes.
  • treating cancer refer generally to a treatment that causes any improvement in a mammal having a cancer, wherein the improvement can be ascribed to treatment with a lipid-nucleic acid particle, nucleic acid or therapeutic gene of the present invention.
  • the improvement can be either subjective or objective. For example, if the mammal is human, the patient may note improved vigor or vitality or decreased pain as subjective symptoms of improvement or response to therapy. Alternatively, the clinician may notice a decrease in tumor size or tumor burden based on physical examination, laboratory parameters, tumor markers or radiographic findings.
  • the present invention is directed to the treatment of solid or diffuse and/or metastatic cancers which consist of cells transfectable with lipid-nucleic acid particles, and which have a low proportion of necrotic and/or quiescent (ie. non-dividing) cells.
  • solid or diffuse and/or metastatic cancers consist of cells transfectable with lipid-nucleic acid particles, and which have a low proportion of necrotic and/or quiescent (ie. non-dividing) cells.
  • necrotic and/or quiescent cells ie. non-dividing cells.
  • examples include, but are not limited to, ovarian, colon, and lung cancers, gliomas and melanomas.
  • the following is a partial list of tumor models used in embodiments of the invention (they may be transformed in vitro and in vivo):
  • inhibiting cell growth or “inhibiting tumor growth” generally means that the size, mass, or rate of increase in mass, size, number and or the metabolism of treated cells and or tumors is lower as a result of treatment than that of nontreated cells and/or tumors.
  • the growth of a cell line or tumor is said to be “inhibited” by a treatment if, when assayed by means such as radioisotope incorporation into the cells, the treated cells increase in number at a rate that is less than the proliferation rate of untreated control cells, and preferably at a rate that is less than about 50% of the untreated cell proliferation rate. More preferably, the growth rate is inhibited by at least 80%. Most preferably, growth is reversed (i.e., the tumor gets smaller).
  • the growth of a cell line is said to be "inhibited” if the treated cells give rise to less than the number of colonies that grow from a like number of untreated cells.
  • the number of colonies from treated cells is less than about 70% of the number from untreated cells. More preferably, the number of colonies is decreased by at least 50%.
  • “Inhibition of cell growth” also encompasses zero growth and, most importantly, consequent death of the tumor cells and eradication of the tumor. When measured in vivo, “inhibition of tumor growth” encompasses fewer or smaller tumors (for example, smaller diameter) as compared to control animals or untreated patients.
  • Inhibition can be evaluated by any accepted method of measuring whether growth or size of the tumor and/or increase in the number of cancerous or tumor cells has been slowed, stopped or reversed. This includes direct observation and indirect evaluation such as subjective symptoms or objective signs.
  • the clinician may notice a decrease in rumor size or tumor burden (number of tumors) based on physical examination, laboratory parameters, tumor markers or radiographic findings.
  • the patient may note improved vigor or vitality or decreased pain as subjective symptoms of improvement or response to therapy.
  • Some laboratory signs that the clinician may observe as an indication of response to therapy include normalization of tests such as white blood cell count, red blood cell count, platelet count, erythrocyte sedimentation rate, and various enzyme levels such as transaminases and hydrogenases. Additionally, the clinician may observe a decrease in a detectable tumor marker such as prostatic specific antigen (PSA) or chorio embryonic antigen (CEA). Alternatively, other tests can be used to evaluate objective improvement such as sonograms, computerized axial tomography scans, nuclear magnetic resonance scans and positron emission testing.
  • PSA prostatic specific antigen
  • CEA chorio embryonic antigen
  • the present invention relates to the surprising discovery that expression of a therapeutic nucleic acid, such as an expressible nucleic acid or gene, is improved using combination treatment with a cell cycle blocker.
  • a therapeutic nucleic acid such as an expressible nucleic acid or gene
  • pretreatment with a cell cycle blocker will cause a greater proportion of cells at the treatment site to synchronize at the G2/M or M phases where the nuclear membrane is dissolved, whereupon exogenous nucleic acids in the cytosol will be captured within the newly forming nuclear membrane immediately after the nuclear division step.
  • the exogenous nucleic acids spend a reduced amount of time outside of the nucleus before they are transcribed.
  • the goal of pretreatment with a cell cycle blocker is to cause a greater proportion of cells at the treatment site (i.e., tumor or other tissue) to be synchronized at the G2/M or M phases.
  • the methods of the present invention will preferably be optimized for each different combination of drug, nucleic acid delivery mechanism and treatment site.
  • the key to the optimization is selection of 1) the proper dosage of the cell cycle blocker (e.g. , drug or radiation) and of the therapeutic gene; and 2) the proper interval between administration of the cell cycle blocker and administration of the therapeutic gene.
  • the "treatment site" component can be a disease indication, such as a neoplasia, or an organ which will benefit from transformation (such as the spleen) to which the drug and the nucleic acid can be administered separately, i.e., by themselves.
  • the optimization relates to the timing and dosages of these separate administrations.
  • Drug accumulation at the treatment site in order to initiate the cell cycle block is governed by the properties of the composition, such as its circulation lifetime, uptake rate, etc. This lead time will influence the total time required to obtain maximum synchronization of cells.
  • Exemplary cell cycle blockers are set out in Table I, infra. Radiation is delivered instantaneously, but the period required for the cells to accumulate at G2/M can be determined by a simple time course experiment;
  • nucleic acid Direct injection to the treatment site presents nucleic acid immediately, although uptake by cells can be accelerated or delayed depending on the carrier (cationic lipid, other transfection additives, buffer alone, etc.).
  • Nucleic acids administered locally or regionally such as viral delivery or cationic complexes
  • systemically such as with intravenously delivered fully lipid encapsulated SPLPs described herein
  • a nucleic acid with a long lag time between administration and accumulation may have to be administered at the same time as the cell cycle blocker or, indeed, in advance of the cell cycle blocker.
  • a heterogenous tumor can be treated first with non-cell cycle specific agents (e.g., cyclophosphamide and doxorubicin) to recruit slowly dividing cells into active DNA synthesis; followed by a cell cycle specific drug, such as methotrexate and 5-FU. The drug cycle is repeated at regular intervals.
  • non-cell cycle specific agents e.g., cyclophosphamide and doxorubicin
  • Two or more non-cross-resistant drugs can be used simultaneously to avoid selection of resistant tumor cells. Double resistant mutants may arise by sequential chemotherapy.
  • the treatment site for the combination therapy when applied in vivo, refers to the site of a disease or disorder which when directly treated by a therapeutic nucleic acid results in a therapeutic benefit for the patient; alternatively, the treatment site can be an organ or normal cell of the body which when directly treated by a therapeutic nucleic acid results in a therapeutic benefit for the patient. Used in this way, therapeutic benefit refers to an objective or subjective evaluation of a patient of improvement in health.
  • Treatment sites that are not disease sites can include organs such as the spleen, liver, lung or kidney.
  • Useful treatment sites can also include cells of the immune system, tissues with secretory ability, tissues with unique transcription promoters that are highly specific for promoter sequences on the plasmid construct, and the like.
  • Another important aspect of the invention is that dosages of cell cycle blocking agent will be carefully optimized for each treatment combination and disease indication.
  • the methods of the invention allow for the use of either a therapeutic or a subtherapeutic dose of the cell cycle blocker.
  • a therapeutic dose falls in the window of dose ranges below unacceptable toxicity and above detectable therapeutic efficacy.
  • a subtherapeutic dose is one which is insufficient to obtain a detectable therapeutic effect.
  • Either dosage is acceptable for use in the methods of the present invention provided the dosage effectively synchronizes cells at the treatment site. Cells that are effectively synchronized using either a therapeutic or a subtherapeutic dosage are suitable for enhanced transformation.
  • the cells at the tumor site fall into one of the following three categories: (1) those exposed to a cell killing amount of drug; (2) those exposed to a cell cycle blocking amount of drug (which block is removed once the drug is metabolized); and (3) cells which do not contact sufficient drug to be affected. It is the cells in the second category that the subsequent treatment with the therapeutic nucleic acid will enhance, given that the cells in the first category are killed and the cells in the third category are not synchronized. If a subtherapeutic dosage of vincristine were employed in the same model, only two cell populations would result, i.e., (2) and (3).
  • cells in the second category will demonstrate enhanced transformation with subsequently administered therapeutic nucleic acids.
  • the methods of the invention therefore, allows for the use of either a therapeutic or a subtherapeutic dosage of cell blocking agent. Further, the methods of the present invention allow for the use of new drugs that were previously unacceptable because of high toxicity at therapeutic levels.
  • quiescent cells which are not amenable to cell cycle modulators or lipid transfection reagents. However, if the cell cycle modulating agent arrests cells irreversibly leading to localized regions of cell death, the previously quiescent cells surrounding these regions now begin to divide and repopulate the regions of cell death. Thus, the repopuiating cells are now amenable to cell cycle modulation for improvement in transfection.
  • Cisplatin S (Unknown) causes intra/inter strand cross linking of DNA
  • Colchicine (Unknown) Prevents endosome/lysosome fusion
  • DTIC Dacarbazine S . purine analogue/alkylating agent Doxorubicin ( Adriamycin) S Binds to DNA/inhibits protein synthesis
  • Taxol (Paclitaxel/Docetaxel) G2/M Prevents depolymerization of microtubules Vinblastine G2/M S and G2/M (inhibits cell energy mechanism)
  • Vincristine G2/M prevents microtubule formation
  • Techniques for in vivo delivery of foreign therapeutic genes can be divided into two classes: (1) those preferred for local or regional delivery (i.e., by inhalation, or direct injection), and (2) those preferred for systemic delivery (i.e., intravenous delivery). According to the methods of the instant invention, either technique can be enhanced by pretreating the cells at the treatment site with a cell cycle blocking agent.
  • the cloning and expression of natural or synthetic nucleic acids is typically achieved by operably linking a nucleic acid of interest to a promoter (which is either constitutive or inducible), incorporating the construct into an expression vector and introducing the vector into a suitable host cell.
  • Typical vectors contain transcription and translation terminators, transcription and translation initiation sequences and promoters useful for regulation of the expression of the particular nucleic acid.
  • the vectors optionally comprise generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in eukaryotes, prokaryotes or both, (e.g., shuttle vectors) and selection markers for both prokaryotic and eukaryotic systems.
  • Vectors are suitable for replication and integration in prokaryotes, eukaryotes or preferably both. See, Giliman and Smith, Gene, 8:81-97 (1979); Roberts, et al., Nature, 328:731-734 (1987); Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology 152, Academic Press, Inc., San Diego, CA (Berger); Sambrook, et al. (1989), Molecular Cloning - A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook); and F.M.
  • a number of vector systems can be used to express the nucleic acids used in the methods of the present invention. These include plasmids, cosmids and a number of viral vectors, including retroviral vectors, vaccinia vectors, lentiviral vectors, herpes simplex vectors, Sindbis/semliki forest viruses, adenoviral vectors, and adeno-associated viral (AAV) vectors.
  • retroviral vectors including retroviral vectors, vaccinia vectors, lentiviral vectors, herpes simplex vectors, Sindbis/semliki forest viruses, adenoviral vectors, and adeno-associated viral (AAV) vectors.
  • retroviral vectors including retroviral vectors, vaccinia vectors, lentiviral vectors, herpes simplex vectors, Sindbis/semliki forest viruses, adenoviral vectors, and adeno-associated viral (AAV) vectors.
  • AAV a
  • Optimal viral delivery systems are characterized by: (1) broad host range; (2) high titer/ ⁇ g DNA; (3) stable expression; (4) non-toxic to host cells; (5) no replication in host cells; (6) ideally no viral gene expression; (7) stable transmission to daughter cells; (8) high rescue yield; and (9) lack of subsequent replication-competent virus that may interfere with subsequent analysis.
  • Choice of vector may depend on the intended application. Preparation of retroviral vectors and their uses are described in many publications including European Patent Application 0 178 220, U.S. Patent No. 4,405,712; Gilboa, Biotechniques, 4:504-512 (1986), Mann, et al, Cell, 33:153-159 (1983); Cone and Mulligan, Proc. Natl. Acad.
  • SPLPs Stable Plasmid-Lipid Particles
  • a preferred method for transforming cells involves the use of lipid-nucleic acid particles. Preferred compositions and methods of making such particles are generally described in U.S. Patent No. 5,705,385 and U.S. Patent Applications Serial Nos. 08/485,458, 08/660,025, 08/484,282, 08/316,399, PCT Publication No. WO 9640964, and Provisional Patent Application Serial No. 60/073,598, all of which are assigned to the assignee of the instant invention and are incorporated herein by reference.
  • a steric barrier compound such as ATTA- lipids; polyethylene glycol (PEG)-lipid derivatives (e.g., PEG-ceramides) or ganglioside GM I -modified lipids, in the lipid-nucleic acid particles used in the methods of the present invention. Addition of such components prevents particle aggregation and provides a means for increasing the circulation lifetime and increasing the delivery of the lipid-nucleic acid particles to the target tissues of interest.
  • the concentration of the steric barrier compound e.g., ATTA-lipid, PEG-ceramide or G i-modified lipids
  • concentration of the steric barrier compound e.g., ATTA-lipid, PEG-ceramide or G i-modified lipids
  • the methods of the invention are also useful for enhanced efficiency of delivery of a therapeutic gene to cells in vitro. It is expected that those of skill in the art are knowledgeable in the numerous expression systems available for expression of DNA. No attempt to describe in detail the various methods known for the expression of proteins in prokaryotes or eukaryotes is made here. There are several well-known methods of introducing nucleic acids into bacterial and animal cells, any of which may be used in the present invention.
  • Transformation of tumor cells is also accomplished by injection of naked DNA directly into a target cell or target tumor cell mass (see, U.S. Patents Nos. 5,580,859 and 5,589,466, both of which issued to Feigner, et al).
  • nucleic acids can be to any cell grown in culture, whether of bacterial, plant or animal origin, vertebrate or invertebrate, and of any tissue or type.
  • concentration of nucleic acid varies widely depending on the particular application, but is generally between about 1 ⁇ mol and about 10 mmol.
  • Treatment of the cells with the nucleic acid is generally carried out at physiological temperatures (about 37°C) for periods of time ranging from about 1 to about 48 hours, preferably from about 2 to about 4 hours.
  • the lipid-nucleic acid particles of the present invention can be adsorbed to almost any cell type. Once adsorbed, the particles can either be endocytosed by a portion of the cells, exchange lipids with cell membranes or fuse with the cells. Transfer or incorporation of the nucleic acid portion of the particle can take place via any one of these pathways. In particular, when fusion takes place, the particle membrane is integrated into the cell membrane and the contents of the particle combine with the intracellular fluid. Contact between the cells and the lipid-nucleic acid particles, when carried out in vitro, will take place in a biologically compatible medium.
  • the concentration of particles can vary widely depending on the particular application, but is generally between about 1 ⁇ mol and about 10 mmol.
  • Treatment of the cells with the lipid-nucleic acid particles will generally be carried out at physiological temperatures (about 37°C) for periods of time ranging from about 1 to about 6 hours and, more preferably, from about 2 to about 4 hours.
  • the delivery of nucleic acids can be to any cell grown in culture, whether of plant or animal origin, vertebrate or invertebrate, and of any tissue or type.
  • the cells will be animal cells, more preferably mammalian cells, and most preferably human cells.
  • a lipid-nucleic acid particle suspension is added to 60-80% confluent plated cells having a cell density of from about 10 " ' to about 10 cells/mL and, more preferably, from about 2 x 10 cells/mL.
  • the concentration of the suspension added to the cells is preferably from about 0.01 to 0.2 ⁇ g/mL and, more preferably, about 0.1 ⁇ g/mL.
  • nucleic acids for example. SPLPs
  • a physiologically-acceptable carrier such as physiological saline or phosphate buffer
  • suitable carriers include, e.g., water, buffered water, 0.4% saline, 0.3% glycine and the like, including glycoproteins for enhanced stability, such as albumin, lipoprotein, globulin, etc.
  • the resulting aqueous solutions may be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.
  • the particle suspension may include lipid-protective agents which protect lipids against free-radical and lipid-peroxidative damages on storage. Lipophilic free-radical quenchers, such as alphatocopherol and water-soluble iron-specific chelators, such as ferrioxamine, are suitable.
  • Formulations suitable for administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • the formulations of packaged nucleic acid can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials.
  • Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • Lipid-nucleic acid particles can be incorporated into a broad range of topical dosage forms including, but not limited to, gels, oils, emulsions and the like.
  • the suspension containing the lipid-nucleic acid particles can be formulated and administered as topical creams, pastes, ointments, gels, lotions and the like.
  • the concentration of particles in the pharmaceutical formulations can vary widely, i.e., from less than about 0.05%, usually at or at least about 2-5% to as much as 10 to 30% by weight and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected. For example, the concentration may be increased to lower the fluid load associated with treatment.
  • particles composed of irritating lipids may be diluted to low concentrations to lessen inflammation at the site of administration.
  • the amount of particles administered will depend upon the particular label used, the disease state being diagnosed and the judgment of the clinician, but will generally be between about 0.01 and about 50 mg per kilogram of body weight, preferably between about 0.1 and about 5 mg/kg of body weight.
  • compositions that comprise the cell cycle synchronizers and/or nucleic acids of the invention are preferably administered parentally, i.e., intraarticularly, intravenously, intraperitoneally, subcutaneously, or intramuscularly. More preferably, the pharmaceutical compositions are systemically administered intravenously or intraperitoneally by a bolus injection.
  • parentally i.e., intraarticularly, intravenously, intraperitoneally, subcutaneously, or intramuscularly.
  • the pharmaceutical compositions are systemically administered intravenously or intraperitoneally by a bolus injection.
  • a bolus injection see Stadler, et al., U.S. Patent No. 5,286,634, which is incorporated herein by reference.
  • Intracellular nucleic acid delivery has also been discussed in Straubringer, et al, Methods in Enzymology, Academic Press, New York, 101:512-527 (1983); Mannino, et al, Biotechniques, 6:682-690 (1988); Nicolau, et al, Crit. Rev. Ther. Drug Carrier Sy St., 6:239- 271 (1989), and Behr, Ace. Chem. Res., 26:274-278 (1993). Still other methods of administering therapeutics are described in, for example, Rahman, et al., U.S. Patent No. 3,993,754; Sears, U.S. Patent No. 4,145,410; Papahadjopoulos, et al, U.S.
  • the pharmaceutical preparations may be contacted with the target tissue by direct application of the preparation to the tissue.
  • the application may be made by topical, "open” or “closed” procedures.
  • topical it is meant the direct application of the pharmaceutical preparation to a tissue exposed to the environment, such as the skin, oropharynx, external auditory canal, and the like.
  • Open procedures are those procedures which include incising the skin of a patient and directly visualizing the underlying tissue to which the pharmaceutical preparations are applied. This is generally accomplished by a surgical procedure, such as a thoracotomy to access the lungs, abdominal laparotomy to access abdominal viscera, or other direct surgical approach to the target tissue.
  • "Closed” procedures are invasive procedures in which the internal target tissues are not directly visualized, but accessed via inserting instruments through small wounds in the skin.
  • the preparations may be administered to the peritoneum by needle lavage. Likewise, the preparations may be administered through endoscopic devices.
  • the nucleic acid can also be administered in an aerosol inhaled into the lungs (see, Brigham, et al., Am. J. Sci, 298(4):278-281 (1989)), or by direct injection at the site of disease (see, Culver, Human Gene Therapy, Mary Ann Liebert, Inc., Publishers, New York, pp. 70-71 (1994)).
  • the particles and/or compositions comprising the particles will have a targeting moiety attached to the surface of the particle.
  • targeting moieties e.g., antibodies, proteins
  • lipids such as those used in the present particles
  • Effective doses of the compositions of the present invention will vary depending upon many different factors, including means of administration, target site, physiological state of the patient, and other medicants administered. Thus, treatment dosages will need to be titrated to optimize safety and efficacy.
  • the physician evaluates the particular nucleic acid used; the disease state being diagnosed; the age, weight, and condition of the patient; circulating plasma levels; vector toxicities; progression of the disease; and the production of anti -vector antibodies.
  • the size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular vector.
  • doses ranging from about 10 ng to 1 g and, more preferable, from about 100 ng to 750 mg DNA per human patient are typical.
  • Doses generally range between about 0.01 and about 50 mg per kilogram of body weight when delivered intravenously; preferably between about 0.1 and about 20 mg/kg of body weight or about 10 8 -10 10 or 10 12 particles per injection.
  • the dose equivalent of a naked nucleic acid from a vector is from about 100 ⁇ g to 750 mg for a typical 70 kilogram patient, and doses of vectors which include a retroviral particle are J J
  • the dosage of therapeutic gene will be influenced by the formulation and method of delivery. Naked DNA or lipid-DNA complexes which are not suitable for systemic delivery may be given by direct injection. In this case, a DNA dosage of about 0.1- 100 ⁇ g per injection site is useful; however, toxic amounts of cationic-lipid complexes must be avoided.
  • lipid-DNA particles such as SPLPs
  • a lipid amounts of 50-150 mg/kg are preferred with 100 mg/kg being most preferable.
  • a suitable dosage of SPLPs for a typical 70 kg patient will be approximately 7 g lipid.
  • SPLPs can be formulated with a wide variety of drug to lipid ratios (i.e., ⁇ 1 to 25% by weight DNA), the total DNA administered to the patient can be varied from ⁇ 70 to about 1750 mg DNA. Doses of lipid below the recommended amounts may be used, but they tend to be more rapidly cleared from blood.
  • Those skilled in the art can determine the proper amount of an SPLP of a suitable drug:lipid ratio based on elementary experimentation, using the principles disclosed herein.
  • infusion Prior to infusion, blood samples are obtained and saved for analysis. Between 10 8 and 1 X 10 12 vectors are infused intravenously over 60-200 minutes. Vital signs and oxygen saturation by pulse oximetry are closely monitored. Blood samples are obtained 5 minutes and 1 hour following infusion and saved for subsequent analysis. At the physician's discretion, reinfusion is repeated about every 2 to 3 months for a total of about 4 to 6 treatments in a one year period. After the first treatment, infusions can be performed on an outpatient basis at the discretion of the clinician. If the reinfusion is given as an outpatient, the participant is monitored for at least about 4 and, preferably, about 8 hours following the therapy.
  • a patient undergoing infusion of a vector or transduced cell develops fevers, chills, or muscle aches, he/she receives the appropriate dose of aspirin, ibuprofen or acetaminophen.
  • Patients who experience reactions to the infusion, such as fever, muscle aches, and chills are premedicated 30 minutes prior to the future infusions with either aspirin, acetaminophen or diphenhvdramine.
  • Meperidine is used for more severe chills and muscle aches that do not quickly respond to antipyretics and antihistamines.
  • Vector infusion is slowed or discontinued depending upon the severity of the reaction.
  • In vivo gene transfer using the methods of the present invention can be practiced in a variety of hosts.
  • Preferred hosts include mammalian species, such as humans, nonhuman primates, dogs, cats, cattle, horses, sheep, and the like.
  • the present invention also provides synchronizing compounds (including cell cycle blockers) and lipid-nucleic acid particles in kit form.
  • the kit will typically be comprised of a container which is compartmentalized for holding the various elements of the kit.
  • the kit will contain the compositions of the present inventions, preferably in dehydrated form, with instructions for their rehydration and administration.
  • Improvements in the transformation efficiency that are obtained using the invention of the present application can be determined in a variety of ways known to those skilled in the art. Two ways, discussed below, are (1) improvement in therapeutic benefit as determined by subjective or objective observation of the patient; and (2) improvement in desired phenotypic change at the physiological or molecular level. Improvements in transformation efficiency that result in therapeutic benefit means generally a result where, if the mammal is human, the patient may note improved vigor or vitality or decreased pain as subjective symptoms of improvement or response to therapy. Alternatively, the clinician may notice a decrease in disease or disorder, or the symptoms of disease or disorder, such as reduced rumor size or tumor burden based on physical exam, laboratory parameters, tumor markers, or radiographic findings.
  • Transformation efficiency is also demonstrated by improvement of the phenotype at the molecular level. This improvement may be ascertained by detection and quantification of the presence and expression of nucleic acids and their gene products at the treatment site.
  • the nucleic acids used in the present invention, and their gene products, are detected and quantified by any of a number of means well known to those of skill in the art.
  • a general summary of these means includes analytic biochemical methods, such as spectrophotometry, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography and the like, and various immunological methods, such as fluid or gel precipitin reactions, immunodiffusion (single or double), immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), immuno fluorescent assays and the like.
  • the detection of nucleic acids proceeds by well known methods such as Southern analysis, northern analysis, gel electrophoresis, PCR, radiolabeling, scintillation counting and affinity chromatography.
  • Plasmids are preferably supercoiled, 4000 to 15000 bp in length, encoding genes and enhancer elements, etc. as desired.
  • Cationic lipid, N,N-dioleyl-N,N-dimethyl ammonium chloride (“DODAC”) and monomethoxy polyethylene2000 glycol succinate- (C8:0-ceramide) (“PEG-Cer-C8”) were synthesized at Inex Pharmaceuticals Corp.
  • Dioleyl-phosphatidylethanolamine (DOPE) was supplied by Northern Lipids, Vancouver. Standard dialysis membranes: Spectro/Por 5 regenerated Cellulose (12-14,000 MWCO) was purchased from VWR (Manufactured by Spectrum Medical Industries Inc.).
  • Plasmid (50-400 ⁇ g) is incubated with DODAC in 500 ⁇ L of the prep solution containing 0.2 M OGP in 150 mM NaCl; 5 mM HEPES pH 7.4, for 30 min at room temperature.
  • This mixture is added to a mixture of DOPE and PEG-Cer-C14 or PEG-Cer-C20 or PEG-Cer-C8 in 500 ⁇ L of the same prep solution.
  • the total lipid concentration was either 5 or 10 mg/mL, with the molar ratio of DOPE:DODAC:PEG- Cer being 84:6: 10.
  • the mixture was dialyzed against 150 mM NaCl; 5 mM HEPES (pH 7.4) for 36-48 h with two buffer changes.
  • Nonencapsulated DNA was removed by anion exchange chromatography on DEAE-Sepharose column (1 x 4 cm). Empty liposomes were removed by pooling lipid/DNA samples that co-eluted on the DEAE column on top of a sucrose density gradient in 12.5 mL ultracentrifuge tubes. The gradient was formed with 3 mL each of 10% sucrose, 2.5% sucrose and 1% sucrose in HBS layered consecutively from bottom to top. The gradients were centrifiiged at 36,000 m (160,000 X g) for 2 h at 20°C in a
  • lipid concentration 5.0 mg/mL (or 5.3 mM); plasmid concentration: 200 ⁇ g; initial volume: 1.0 mL; and lipid stock solutions: (in 95:5 benzene :methanol, 2:1 chloroform:methanol or ethanol).
  • DOPE 744 g/mol
  • DODAC 582 g/mol
  • PEG-C8 2515 g/mol
  • 20 mM DOPE DODAC PEG-C8 mg 1.68 1.315 2.005 mole % 42.5 42.5 15 ⁇ mol 2.25 2.25 0.8 ⁇ l 56.2 56.2 40
  • the procedure for making the formulation on a 1 mL scale was as follows: Aliquot lipid stock solutions into a clean, dry test tube and dry to a lipid film using a stream of N 2 gas and then dry under vacuum for at least 2 hours. Add 50 ⁇ L 2M OGP and add 500 ⁇ L of 2X strength dialysis buffer, add 200 ⁇ g of plasmid and mix by vortexing to dissolve the lipid film. Make up to 1.0 mL with sterile deionized H 2 0, mix and allow to incubate approximately 30 min. at room temperature .
  • Formulation 1.2 After DEAE cleaning and sucrose density centrifugation, Formulation 1.2 has a concentration of 200 ⁇ g/mL plasmid and 5 mg/mL total lipid.
  • DODAC cationic lipid
  • Formulations below 30% DODAC are preferably made in 10 mg total lipid.
  • Dialysis buffer can be changed as in Table 2, below:
  • Lipid-plasmid particles with 10-30% DODAC are also useful in the present invention. These can be formulated as described above or as follows.
  • Lipid stock solutions Individual stock solutions of each lipid were dissolved in chloroform/methanol (2:1 v/v) to a final concentration of 2 or 20 mg/mL.
  • OGP solution 1.0 M OGP solution was prepared in MilliQ grade water.
  • Citrate buffer Sodium citrate buffer was used for dialysis to remove detergent from the formulation. The citrate concentrations were varied according to the amount of DODAC. The buffer also contains 150 mM NaCl and 5 mM HEPES at pH 7.4, unless indicated otherwise. In general, a 10X solution was prepared and diluted 1 :10 in MilliQ Plus water for dialysis using a graduated cylinder.
  • lipid/DNA/OGP mixture A typical formulation contained 10 mg of lipid of DODAC/DOPE/PEG-Cer-C8 and 200 ⁇ g DNA. Appropriate amounts of stock solutions containing DODAC, DOPE and PEG-Cer-C8 (normally 15 mol % in this study) were mixed in a glass test tube. If the amount of DODAC is changed, the amount of DOPE is changed to maintain a total of 10 mg lipid. The solvent was first removed under a stream of N 2 gas followed by incubation under vacuum for 3-5 h. To the lipid, 0.2 mL of 1 M OGP was added. The suspension was vortexed until the lipid was totally dissolved and the solution became clear.
  • a 0.2 mL DNA (1 mg/mL) solution containing 200 ⁇ g DNA and 0.6 mL HBS (HEPES buffered saline) or citrate buffer (concentrations designated in Figure 1) were added to a final total volume of 1 mL. If the solution did not become clear, a small amount of OGP (50 ⁇ L) was added. The solution was incubated at room temperature for 1 hr to allow the components to equilibrate. Dialysis: Dialysis tubes were soaked in 60% ethanol (or in distilled water if sterilization was not required) for 30 min. The mixture of DNA/lipid/OGP solution was then transferred to the dialysis tube. The sample was dialyzed for 2 days in 2-4 L citrate buffer (concentration as described in Figure 1) with two changes of buffer daily.
  • HBS HBS buffered saline
  • citrate buffer concentration designated in Figure 1
  • membrane modifying lipids such as cholesterol or DOPE
  • membrane modifying lipids such as cholesterol or DOPE
  • alternative cationic lipids such as DMRIE, DOTAP, DOTMA, DODMA, AL-1, etc.
  • fusogenic components such as pH sensitive lipids, peptides (EALA) or polymers (PEAA); use of targeting agents: use of DNA condensing peptides (i.e., polylysine or spermine) or polymers (i.e., PEI); use of negatively charged lipids, such as phosphatidylserine; or use of alternative PEG-lipid linkers, such as SPDPs or PDPH (disclosed in U.S. Patent Application Serial No. 08/536,584, which is assigned to assignee of the instant invention).
  • fusogenic components such as pH sensitive lipids, peptides (EALA) or polymers (PEAA)
  • targeting agents use of DNA condensing peptides (i.e., polylysine or spermine) or polymers (i.e., PEI); use of negatively charged lipids, such as phosphatidylserine; or use of alternative PEG-lipid linkers, such as SPDPs or PDPH (
  • Formulation 1.4 also known as INEX 303
  • Formulation 1.4 contains DOPE:DODAC:PEG-Cer-C20 (83:7:10) mol %.
  • the synthesis protocol is as follows: Aliquot the lipid stock solutions (in ethanol) into an autoclaved, clean, dry round bottom flask. The solution is dried to a lipid film using a rotavap in a 65 °C water bath and vacuumed overnight. Add HBS with octylglucopyranoside (OGP) to a final OGP concentration of 200 mM. Swirl the mixture to dissolve the lipid film and, if necessary, heat to 37°C to ensure the lipid is fully dissolved. Plasmid DNA is then added (400 ⁇ g /10 mg lipid) to the dissolved lipid films.
  • OGP octylglucopyranoside
  • Nonencapsulated DNA was removed by anion exchange chromatography on a DEAE-Sepharose CL-6B column. Collect the particle suspension as it appears in the eluate, and concentrate using the Amicon diafiltration system (YM 30 membrane). Next, empty liposomes were removed using a sucrose density gradient. The gradient was formed by layering 10% sucrose, 5.0% sucrose and 2.5% sucrose in HBS, pH 7.4. The sample is loaded by floating it on top of the 2.5% sucrose layer and centrifuging at 28,000 ⁇ m for 18 hours at 20°C using a Beckman Optima XL-100K ultracentrifuge and an SW-28 rotor. After centrifugation, remove the lower band with a syringe and needle and pool the samples.
  • the sucrose is removed and the sample is concentrated simultaneously using the Amicon system. Filter sterilize the final volume through a 0.2 micron filter.
  • DNA concentration is analyzed using, for example, Picogreen assays; lipid concentration is analyzed using, for example, HPLC; and particle size is analyzed using, for example, Nicomp analysis.
  • Formulation 1.5 also known as "ethanol method"
  • This method is an alternative high-efficiency formulation of the lipid/nucleic acid particle. This method is disclosed in PCT Patent Publication No. WO 96/40964, the teachings of which are inco ⁇ orated herein by reference. It is, in essence, a preparation of lipid therapeutic nucleic acid particles in organic solvent.
  • PEG-Cer-C20 (or C14) 50 mg/mL (10 mol %) - 67.6 ⁇ L.
  • the lipids are mixed together and the volume is increased to a total volume of 0.400 mL with 100% ethanol.
  • An appropriate volume of 300 mM citrate buffer (pH 3.3) is added to the DNA to a final volume of 600 ⁇ L and pH 3.8.
  • SK-OV-3 cells human ovarian tumor cells, ATCC were cultured in RPMI 1640 media (StemCell Technologies, Vancouver, B.C.) supplemented with 10% fetal bovine serum (Intergen).
  • HeLa-luc cells were stably transfected with a luciferase reporter plasmid and cultured in Dulbecco's modified Eagle medium (StemCell Technologies, Vancouver, B.C.) supplemented with 10% fetal bovine serum and 400 ⁇ g/mL Geneticin (Gibco BRL).
  • an expression vector, pINEX L018 in which the Photinus pyralis luciferase gene (luc+ from Promegaunder the control of the CMV promoter (P. Tarn, unpublished) was used.
  • the HeLa-luc cell line was created by transfection of HeLa cells with a plasmid pINEX L032 containing the P. pyralis luciferase gene (luc+ from Promega) and a neomycin resistance gene (based on pIRESlneo from Clontech); both under the control of the CMV promoter.
  • Supercoiled plasmid DNA was purified using a modified alkaline lysis procedure followed by purification using a CsCl / EtBr gradient (Maniatis, et al, Molecular Cloning, Cold Spring Harbor, New York, 1981, pp. 93-94).
  • Lipoplexes were prepared as follows: All reagents were sterile and cooled to 4°C prior to complex preparation. A solution containing 62 ⁇ M dioleoyldimethylammonium chloride: 1 ,2-sn-dioleoylphosphatidylethanolamine
  • Plasmid containing a therapeutic gene was encapsulated as SPLP using the detergent dialysis method of INEX 351.
  • the SPLP contained 42.5:42.5:15 mol % of DOPE:DODAC: 1-0- [2'-(w-methoxypolyethyleneglycol) succinoyl]-2-N- octoylsphingosine. (PEG-CerCg), at a lipid:DNA ratio of 15:1 (w/w). SPLPs were separated from empty liposomes by centrifugation through a discontinuous sucrose gradient.
  • pINEX L018 an expression vector in which the Photinus pyralis luciferase gene (Promega, Madison WI) is under the control of the CMV promoter.
  • pINEX-TKl 0 an expression vector comprising a pBR322 derived plasmid with CMV promoter linked to a "hyper" HSV-TK gene (see, Black, et al, PNAS USA, 93:3525-3529 (1996)).
  • pINEX-IL-12 is similar to pINEX-TKlO except that the therapeutic gene comprises the IL-12 gene linked to the CMV promoter.
  • SK-OV-3 or HeLa-luc cells were seeded at a density of 30,000 cells per well in a 24 well plate in growth media 24 hours prior to treatment with aphidicolin.
  • Aphidicolin (Sigma) was reconstituted in phosphate buffered saline containing 0.5 % DMSO. Cells were incubated with aphidicolin at a final concentration of 5 ⁇ g/mL in 1 mL of media per well for 16 hours prior to transfection. Transfection was accomplished by the addition of 0.5 ⁇ g of formulated DNA to each well.
  • Luciferase cDNA was cloned into the multiple cloning site of pSFVl-3. Recombinant virus encoding luciferase was prepared according to the protocol described by Liljestrom and Garoff (Maniatis, et al, Molecular Cloning, Cold Spring Harbor, New York, 1981, pp. 93-94). Prior to infection of SK-OV-3 cells, virus particles were activated in 0.5 mg/mL chymotrypsin for 30 minutes at room temperature. Chymotrypsin was inactivated by the addition of aprotinin to a final concentration of 0.67 mg mL. Activated virus at a multiplicity of infection of 0.5 was added directly to the cells in growth media. g. Assessment of Cell Cycle Status
  • the cell cycle status of cultures was determined by flow cytometry of permeabilized cells that were stained with propidium iodide. Prior to harvesting, media was aspirated and the cells were washed twice with 2 mL phosphate buffered saline (PBS). After washing, 2-3 drops of pre-warmed trypsin-EDTA (0.25 % trypsin, 1 mM EDTA; Gibco BRL) was added to each well and the plate was incubated at 37°C until the cells had detached from the plate. Trypsin was inactivated by the addition of 1.5 mL of cold media.
  • PBS phosphate buffered saline
  • the samples were transferred to 6 mL polystyrene tubes (Becton-Dickenson, San Jose, CA, USA) and centrifiiged at 2000 m for 5 min (Beckman GS-6R, rotor GH3.8). Cells were fixed and permeabilized by incubation for 10 min in 70 % methanol (pre-chilled to -70°C ). Samples were then centrifiiged at 2000 ⁇ m for 5 min and the cells were incubated in 0.5 mL of 150 ⁇ g/mL propidium iodide (Molecular Probes, Eugene, OR) in PBS for 30 minutes on ice in the dark prior to analysis by flow cytometry.
  • propidium iodide Molecular Probes, Eugene, OR
  • Plasmid DNA was fluorescently labeled by incubation with YOYO-1 iodide (Molecular Probes. Eugene, OR) at a dye:basepair ratio of 100: 1 for 10 minutes at 4°C. The sample was then dialyzed twice against autoclaved deionized water.
  • Luciferase assays were performed using the Luciferase Assay System kit (Promega, Madison, WI) according to the manufacturer's protocol. Cell samples were washed twice with PBS, lysed by incubation with 200 ⁇ L of lysis buffer (0.1 % triton in 250 mM NaH 2 P0 4 , pH 7.4) for 20 min. 20 ⁇ L of cell lysate was assayed (in duplicate) using a mL3000 microtiter plate luminometer (Dynex Technologies). A standard curve was determined by assaying 20 ⁇ L of serial dilutions of a 1 mg/mL luciferase standard (Boehringer Mannheim).
  • Cell lysate was assayed for protein content using the bicinchoninic acid (BCA) colorimetric method. Briefly, in a 96 well plate, 95 ⁇ L of water and 100 ⁇ L of the Micro BCA protein assay working reagent (Pierce) were added to 5 ⁇ L of cell lysate. The plates were shaken on a rotary platform at 100 ⁇ m for 10 minutes before being incubated at 37°C for 2 hours. Absorbance was monitored at 570 nm (Dynatech MR5000 plate reader) and protein content calculated using a standard curve of varying concentrations ofBSA.
  • BCA bicinchoninic acid
  • Aphidicolin inhibits DNA polymerase ⁇ and ⁇ , the two major polymerases involved in DNA synthesis during the S-phase (see, Ikegami, et al, Nature, 275:458-460 (1978); Wahl, et al, Biochemistry, 25:7821-7827 (1986); Legendre, et al, Pharm. Res., 9:1235-1242 (1992)), and specifically arrests cells in the GJS boundary.
  • the effectiveness of a non-lethal concentration of aphidicholin to arrest SK-OV-3 cells was determined.
  • a sub-confluent asynchronous cell culture contained approx.
  • a clonal HeLa cell line stably transfected with a CMV driven luciferase expression cassette (Song, Inex Pharmaceuticals, unpublished) was analyzed.
  • This cell line, HeLa-luc was engineered to constitutively express the gene encoding luciferase. If aphidicholin treatment down regulates CMV driven transcription or the host cells ability to transport and translate the luciferase mRNA, it is expected that aphidicholin treated HeLa-luc cells would contain less luciferase activity than untreated cells.
  • FIG. 3 shows that treatment with aphidicolin did not significantly affect the level of luciferase gene expression when compared with the untreated control cells.
  • the results from these experiments illustrate that aphidicolin treatment effectively arrests cells in Gi phase of the cell cycle with negligible concomitant effects on the internalization of lipoplexes and processes involved in gene expression. 5.
  • Cells arrested in the G S phase by aphidicolin are not transfected efficiently by lipoplexes.
  • Figure 5 shows the correlation of cell cycle status of a (+/-) culture and the kinetics of luciferase gene expression after incubation with either lipoplexes (Figure 5(A)) or SPLPs ( Figure 5(B)).
  • Incubation with either lipid formulations in (+/+) cultures show extremely low levels of luciferase activity throughout the course of the experiment.
  • In the (+/+) cultures 95% of the cells remained arrested in the Gi phase throughout the course of the experiments (not shown).
  • the (-/-) cultures began to exhibit significant luciferase activity after 6-8 hr of incubation with either lipid formulation. The levels of luciferase activity progressively increased and reached maximal levels at 26 hrs when the experiment was terminated.
  • Figures 6(A) and (B) show the transfection profiles of cultures incubated with lipoplexes or SPLPs, respectively.
  • luciferase expression was observed to increase in (+/-) cultures, beginning at approximately 10 hours after the removal of aphidicolin and reaching a maximum at 12 hours.
  • the (+/-) cultures exhibited 3-fold and 8-fold increases in luciferase expression with lipoplexes and SPLPs, respectively.
  • Analysis of the cell cycle status of the (+/-) cultures indicated that progression of cells from G 2 /M phase into G_ phase had occurred at 10 hours after the removal of aphidicolin ( Figure 6(C), 10 hrs).
  • SFV alphavirus Semliki Forest Virus
  • Alphaviruses are enveloped viruses that contain a positive sense strand RNA genome.
  • the genomic RNA serves as a substrate for cytoplasmic translation and replication upon infection. Infection with replication defective SFV vectors is therefore not dependent on nuclear delivery of the genetic material.
  • the virus, SFV-luc used in this study contained the gene encoding luciferase in place of the viral structural genes.
  • the known therapeutic dosage of liposomal vincristine is 2 mg/kg. Doses below this level are considered subtherapeutic.
  • a 200 ⁇ L dosage of OncoTCS administered to an approximately 25 g mouse is approximately 0.5 mg/kg vincristine sulfate and 10 mg/kg lipid. These liposomes provide extended release of vincristine. Vincristine sulfate in the free form may also be used in the methods of the present invention.
  • mice 25 C57 mice are seeded intraperitoneally with about 100,000 B16 cells. On day 7, 12 mice are injected i.v. via the tail vein with about 0.5 mg/kg of OncoTCS
  • INEX 324 TCS containing luciferase plasmid pINEX L0128
  • Tumors are harvested 6, 12, 24 and 48 hours later, fast frozen, and stored at - 70°C until analyzed for luciferase activity. The frozen organs are then thawed at room temperature. 600 microliters of IX cell culture lysis buffer (Promega) is added and the samples are homogenized in a FASTPREP homogenizer for 20 sec.
  • This example demonstrates the synchronization effect caused by the cell cycle blocker vincristine at a tumor site.
  • mice carrying MCA 207 tumors were injected at time 0 i.v. via tail vein with either empty OncoTCS (i.e., empty SM/chol liposomes; 10 mg/kg lipids in 200 ⁇ L total solution) or OncoTCS (200 ⁇ L dosage: 0.5 mg/kg vincristine sulfate).
  • OncoTCS i.e., empty SM/chol liposomes; 10 mg/kg lipids in 200 ⁇ L total solution
  • OncoTCS 200 ⁇ L dosage: 0.5 mg/kg vincristine sulfate
  • mice were euthanized either at 24 h or at 72 h after treatment. Tumors were excised and evaluated as follows. Freshly harvested tumors were homogenized in 7 mL of media into single cells using a Dounce homogenizer (10-15 strokes). The cell suspension was carefully decanted into a 15 mL Falcon tube ensuring that non- homogenized tissue was not collected. The cells were centrifuged for 5 min at 1000 g (4°C). The cell pellet was thoroughly resuspended in 2 mL 70% methanol and stored at 70°C for 20 min. The cells were then pelleted by centrifugation for 5 min at 1000 g (4°C).
  • the cell pellet was resuspended in 1 mL 100 ⁇ g/mL propidium iodide and stored at 4°C for 15 min before analysis by flow cytometry.
  • Flow cytometry analysis was performed on FACSort (Becton-Dickinson) and cells analyzed on FL2 channel utilizing doublet discrimination mode.
  • Figure 9 Data in Figure 9 is presented as a histogram showing FL2-Area (x-axis) vs. number of cells (y-axis).
  • n normal somatic cell DNA content
  • 2n cells accumulated at phase immediately prior to cell division.
  • Figure 9A demonstrates that mice MCA 207 tumors treated with empty OncoTCS cells evidence very few cells ( ⁇ 5%) at the 2n phase after 24h, essentially the same as the negative control. Cell cycle distribution is approximately 85%, 5% and 10% in G0/G1 , S and G2/M phases, respectively.
  • Figure 9B demonstrates a significant accumulation at 24 h after OncoTCS administration of tumor cells at the 2n phase (45-55%.
  • FIG. 9C demonstrates that by 48 h after vincristine treatment, most cells have progressed through the cell cycle block and are returning to their normal pretreatment distribution. The results demonstrate that a subtherapeutic amount of vincristine results in effective transient synchronization of cells at the 2n phase, with a significant accumulation at 24h. The results suggest that accumulation of the therapeutic nucleic acid at the tumor site in the period around 24 h-48 h after OncoTCS administration would provide the greatest therapeutic advantage.
  • This example demonstrates that pretreatment of mice bearing subcutaneous tumors with the vinca alkaloid drug vincristine improves transfection at the tumor site of a reporter gene construct pINEX L018 encapsulated in an INEX 351 lipid formulation administered by direct intra-tumoral injection.
  • This example uses mice bearing MCA 207 fibrosarcoma tumor cells (provided by S. Rosenberg, National Cancer Institute, Frederick/Bethesda, MD), but the data suggests that any standard tumor model, including Lewis Lung or U87 tumors will respond to the methods of the present invention.
  • C57 BL/6 mice are seeded subcutaneously with MCA 207 tumor cells. Tumor seeding is performed by standard techniques.
  • mice were seeded subcutaneously on the hip flank with 100,000 MCA-207 fibrosarcoma tumor cells by subcutaneous injection on day zero.
  • the tumor cells had been cultivated and prepared according to standard techniques using RPMI media with 10% Fetal Bovine Serum (see, for example, Current Protocols in Molecular Biology, Eds. Ausubel, F.M., et al.)
  • mice were injected i.v. via tail vein with approximately 0.5 mg/kg of OncoTCS (200 ⁇ L) (subtherapeutic amount of vincristine). Vincristine sulfate in the free form may also be used in known therapeutic dosages.
  • Luciferase assay The frozen tissues are thawed to room temperature. 600-800 ⁇ L of IX cell culture lysis buffer (Promega) is added and the samples are homogenized in the FASTPREP homogenizer for 20 sec at speed 4.0. The samples are centrifiiged for 2 min at 10,000 g and 20 ⁇ L of the supernatant is assayed for luciferase using standard techniques.
  • Figure 10 demonstrates the effect of pretreatment of OncoTCS (i.v., 0.5 mg/kg) on transfection and expression of pINEX L018 at an MCA 207 tumor site using an INEX 351 formulation (i.t., 2 ⁇ g DNA). Tumors were harvested either 12 h or 24 h after administration of the plasmid. Luciferase activity calibrated per gram of tumor mass is approximately 10 times higher after pretreatment with OncoTCS.
  • Figure 11 The results of Figure 11 were obtained using U87 tumor bearing mice.
  • U87 tumor cells (ATCC #HTB- 14). These cells were prepared and seeded subcutaneously on the hip flank of C57 bl/6 mice according to standard techniques described above.
  • mice were seeded subcutaneously on the hip flank with 100,000 MCA-207 fibrosarcoma tumor cells by intra-dermal injection on day zero.
  • the tumor cells had been cultivated and prepared according to standard techniques using RPMI media with 10% Fetal Bovine Serum (see, for example, Current Protocols in Molecular Biology, Eds. Ausubel, F.M., et al.)
  • mice were injected i.v. via tail vein with approx. 0.5 mg/kg of OncoTCS (200 ⁇ L). Vincristine sulfate in the free form may also be used in known therapeutic dosages. Control mice were injected with empty SM/chol liposomes.
  • mice were injected i.v. via tail vein with INEX 324 (100 ⁇ g DNA pINEX-11-12 with about 500 ⁇ g lipid all in about 200 ⁇ L total volume).
  • Control mice received HBS 8 hr after TCS administration, mice were euthanized and the spleens harvested and analyzed for luciferase activity. Harvested tumors were fast frozen and stored at -70°C until analyzed for luciferase activity.
  • Luciferase assay The frozen tissues are thawed to room temperature. 600- 800 ⁇ L of IX cell culture lysis buffer (Promega) is added and the samples are homogenized in the FASTPREP homogenizer for 20 sec at speed 4.0. The samples are centrifiiged for 2 min at 10,000 g and 20 ⁇ L of the supernatant is assayed for luciferase using standard techniques.
  • Figure 12 demonstrates an enhanced level (up to 100-fold) of luciferase activity at the spleen after pretreatment with OncoTCS, according to this example.
  • the level of gene expression is dependent on the interval between OncoTCS pretreatment and INEX 324 administration with maximum difference ( ⁇ 100-fold) observed at 36 hr interval.
  • This experiment confirms a principle of the invention that selection of the appropriate interval between administration of the cell cycle blocking agent and administration of a nucleic acid can leading to greatly improved expression of an expressible gene.
  • This experiment also demonstrates that improved transfection and expression of nucleic acids following pretreatment with a cell cycle blocking agent can be obtained at cells other than tumor cells.
  • mice C57 BL/6 mice are seeded subcutaneously with MCA 207 tumor cells. Tumor seeding is performed by standard techniques. Mice were seeded subcutaneously on the hip flank with 100,000 MCA-207 fibrosarcoma tumor cells by sub-cutaneous injection on day zero. The tumor cells had been cultivated and prepared according to standard techniques using RPMI media with 10% Fetal Bovine Serum (see, for example, Current Protocols in Molecular Biology, Eds. Ausubel, F.M., et al.)
  • mice were injected i.v. via tail vein with approximately 0.5 mg/kg of OncoTCS (200 ⁇ L).
  • mice were injected i.v. via tail vein with INEX 324 (100 ⁇ g DNA pINEX-IL-12 with about 500 ⁇ g lipid all in about 200 ⁇ L total volume).
  • Control mice received HBS. Mice are observed and tumor sizes are recorded daily employing standard measurement techniques using calipers.
  • Results demonstrate that a subtherapeutic dose of vincristine greatly enhances the therapeutic effect of the pINEX-IL-12 construct in INEX 324 formulation. Tumor growth is inhibited and regression in tumor size is visible.
  • This experiment demonstrates the use of the invention to treat cancer by causing transfection at sites other than the tumor itself.
  • the mechanism may result from selective transfection of normal somatic cells, such as in the spleen, using genes encoding immune stimulatory agents such as interleukin 12 (IL-12), which cause an effect at a distal site.
  • IL-12 interleukin 12
  • This example demonstrates that the in vivo therapeutic efficacy of an expressible nucleic acid in an SPLP administered by systemic (i.e., intravenous) injection can be improved by pretreatment of the subject with a conventional drug.
  • the conventional drug is a subtherapeutic amount of vincristine;
  • the SPLP is the same HSV- TK ganciclovir suicide gene system disclosed in U.S. Provisional Patent Application Serial No. 60/086,917, which is entitled "Systemic Delivery of Serum Stable Plasmid Lipid Particles For Cancer Therapy," and which is assigned to the assignee of the instant invention and is inco ⁇ orated herein by reference.
  • INEX 303 containing the HSV-TK gene plasmid construct demonstrated therapeutic efficacy against tumors when administered intravenously in conjunction with the prodrug ganciclovir.
  • This example demonstrates that the previous work can be improved by pretreatment with a cell cycle blocking agent.
  • C57 BL/6 mice are seeded subcutaneously with MCA 207 tumor cells.
  • Tumor seeding is performed by standard techniques. Mice are seeded subcutaneously on the hip flank with 100,000 MCA-207 fibrosarcoma tumor cells by intra-dermal injection on day zero. The tumor cells are cultivated and prepared according to standard techniques using RPMI media with 10% Fetal Bovine Serum (see, for example, Current Protocols in Molecular Biology, Eds. Ausubel, F.M., et al.)
  • mice are injected i.v. via tail vein with approximately 0.5 mg/kg of OncoTCS (200 ⁇ L), as described in the other examples.
  • mice are injected i.v. via tail vein with INEX 303 containing pINEX-TKlO prepared as described herein (100 ⁇ g DNA with about 500 ⁇ g lipid all in about 200 ⁇ L total volume). Control mice receive HBS.
  • 1 mg ganciclovir which is in 200 ⁇ L PBS (approximately 50 mg/kg; commercially available from Hoffman La Roche) is administered by intraperitoneal injection 12 h and 24 h after SPLP administration. Mice are observed and tumor sizes are recorded daily employing standard measurement techniques using calipers.
  • Results demonstrate pretreatment of subjects with a subtherapeutic level of cell cycle blocker inhibits growth of tumors and promotes tumor regression to a significantly greater degree than simply using the HSV-TK/ganciclovir system alone. Additionally, the subtherapeutic dose of vincristine reduces its toxic side effects and makes treatment more tolerable for patients. e. In vivo combination therapy employing expressible nucleic acids and conventional drugs #3
  • mice (Harlan Sprague Dawley or Charles River Laboratory) are seeded subcutaneously with Neuro2a tumor cells (neurological tumor # CLL 131). Tumor cells were prepared and seeded using standard techniques. Mice were seeded on the hip flank with 1.5 million tumor cells by subcutaneous injection on day zero. The tumor cells had been cultivated and prepared according to standard techniques using RPMI media with 10% Fetal Bovine Serum (see, for example, Current Protocols in Molecular Biology, Eds. Ausubel, F.M., et al). Experiments were performed starting on days 7-10, when tumors had developed to useable sizes (30-100 mg).
  • mice were injected i.v. via tail vein with INEX 303 (100 ⁇ g DNA pINEX LOl 8 with about 500 ⁇ g lipid all in about 200 ⁇ L total volume).
  • Control mice received HBS (buffer only).
  • mice were euthanized and sacrificed 48 hours after plasmid administration. Tumors were immediately removed from the mice and flash frozen and stored until luciferase expression activity in the tumor could be measured according to standard techniques.
  • Figure 14 sets out the schedule of vincristine administrations.
  • mice were injected i.v. via tail vein with approximately 0.5 mg/kg of OncoTCS (200 ⁇ Ls).
  • the schedule sets out the OncoTCS (i.e., vine) administration schedule.
  • OncoTCS was administered either before or after the plasmid, and two doses of OncoTCS were sometimes administered.
  • the relative timing of administrations reflects the strategy of optimizing the delivery of the Inex 303 to the tumor site, and coordinating that delivery with the release of cells from the cell cycle blocker.
  • Inex 303 is a relatively long circulating formulation, so accumulation of large doses at the tumor site requires longer time periods.
  • Results in Figure 15 demonstrate an extraordinar sensitivity of the tumors to the relative timing of OncoTCS and SPLP administrations.
  • Groups A and C demonstrate that when SPLP is administered in advance of OncoTCS, expression is about three times greater if the tumors are exposed to vincristine for an extra 8 hours (i.e., 32 h instead of 24 h).
  • Groups E and F demonstrate that this 8 hour effect is not significant when the OncoTCS is administered 64-72 h before tumor harvest. It is suggested from this data that a critical parameter for improved gene expression, when the timing of SPLP administration to tumor harvest is fixed at 48 hours, is the total amount of time from OncoTCS administration to harvest.
  • Figure 15 also shows that two doses of OncoTCS in advance of SPLP administration result in an approximately 4-fold increase in luciferase expression at the tumor compared to controls (Groups D and G vs. Group E). It is throught that the great improvement in luciferase expression may be attributed to the extra dose of OncoTCS, which is approaching therapeutic levels.
  • Figure 16 demonstrates, in a repeat experiment, that Group C (minus 32 h) shows greater transfection than a minus 16 h time point.
  • nucleic acids can also be achieved using an alternative method of synchronizing cells, namely, X-ray therapy. Again, proper selection of the interval between the administration of the cell cycle blocking agent and the administration of the nucleic acid is useful to obtain greatest improvement in expression levels.
  • C57 BL/6 mice are seeded intraperitoneally with B16 tumor cells (CRL 6322). Tumor seeding was performed by standard techniques. Mice were seeded intraperitoneally with 100,000 B 16 tumor cells by injection on day zero. The tumor cells had been cultivated and prepared according to standard techniques using RPMI media with 10% Fetal Bovine Serum (see, for example, Current Protocols in Molecular Biology, Eds. Ausubel, F.M., et al.)

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  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

La présente invention concerne des méthodes permettant d'améliorer l'efficacité de la transformation de cellules à cycle. Ces méthodes consistent à synchroniser des cellules dans un premier stade du cycle cellulaire, puis à transformer ces cellules, dans un second stade du cycle cellulaire en l'espace d'environ un cycle cellulaire du premier stade, avec un acide nucléique obtenu par génie génétique codant un produit génique voulu. L'invention concerne également une thérapie anticancéreuse et, en particulier, des méthodes permettant de transformer efficacement des cellules cancéreuses avec des acides nucléiques codant des produits géniques inhibant la croissance de cellules cancéreuses.
PCT/CA1999/000371 1998-04-22 1999-04-22 Traitement associe utilisant des acides nucleiques et des medicaments classiques WO1999054445A2 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP99917711A EP1082419A2 (fr) 1998-04-22 1999-04-22 Traitement associe utilisant des acides nucleiques et des medicaments classiques
JP2000544777A JP2002512258A (ja) 1998-04-22 1999-04-22 核酸と従来の薬物とを使用する併用療法
AU35912/99A AU762986B2 (en) 1998-04-22 1999-04-22 Combination therapy using nucleic acids and conventional drugs
CA002325561A CA2325561A1 (fr) 1998-04-22 1999-04-22 Traitement associe utilisant des acides nucleiques et des medicaments classiques

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US8266598P 1998-04-22 1998-04-22
US60/082,665 1998-04-22
US11163598P 1998-12-09 1998-12-09
US60/111,635 1998-12-09
US09/295,663 1999-04-21
US09/295,663 US6841537B1 (en) 1998-04-22 1999-04-21 Combination therapy using nucleic acids and conventional drugs

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WO1999054445A2 true WO1999054445A2 (fr) 1999-10-28
WO1999054445A3 WO1999054445A3 (fr) 1999-12-09

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JP (1) JP2002512258A (fr)
AU (1) AU762986B2 (fr)
CA (1) CA2325561A1 (fr)
WO (1) WO1999054445A2 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003093469A2 (fr) * 2002-05-01 2003-11-13 Chromos Molecular Systems, Inc. Procedes d'administration de molecules d'acides nucleiques dans des cellules et evaluation associee
US6849599B2 (en) 2000-03-08 2005-02-01 Rhode Island Hospital Combination drug therapy
US6936469B2 (en) 2001-03-22 2005-08-30 Chromos Molecular Systems Inc. Methods for delivering nucleic acid molecules into cells and assessment thereof
US7294511B2 (en) 2001-03-22 2007-11-13 Chromos Molecular Systems, Inc. Methods for delivering nucleic acid molecules into cells and assessment thereof

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GB1548621A (en) * 1975-07-10 1979-07-18 Lilly Co Eli Nhydrovinca derivatives
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US5688773A (en) * 1994-08-17 1997-11-18 The General Hospital Corporation Method of selectively destroying neoplastic cells

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Publication number Priority date Publication date Assignee Title
GB1548621A (en) * 1975-07-10 1979-07-18 Lilly Co Eli Nhydrovinca derivatives
WO1994000095A2 (fr) * 1992-06-24 1994-01-06 Cortex Pharmaceuticals, Inc. Emploi d'inhibiteurs de calpaine dans l'inhibition et le traitement d'etats medicaux associes a une activite de calpaine accrue
WO1995008994A1 (fr) * 1993-09-29 1995-04-06 Indiana University Foundation Combinaison de taxol et de tiazofurine dirigee contre les neoplasmes
US5688773A (en) * 1994-08-17 1997-11-18 The General Hospital Corporation Method of selectively destroying neoplastic cells

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SON K ET AL: "EXPOSURE OF HUMAN OVARIAN CARCINOMA TO CISPLATIN TRANSIENTLY SENSITIZES THE TUMOR CELLS FOR LIPOSOME-MEDIATED GENE TRANSFER" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol. 91, 1 December 1994 (1994-12-01), pages 12669-12672, XP002039693 ISSN: 0027-8424 cited in the application *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6849599B2 (en) 2000-03-08 2005-02-01 Rhode Island Hospital Combination drug therapy
US6936469B2 (en) 2001-03-22 2005-08-30 Chromos Molecular Systems Inc. Methods for delivering nucleic acid molecules into cells and assessment thereof
US7294511B2 (en) 2001-03-22 2007-11-13 Chromos Molecular Systems, Inc. Methods for delivering nucleic acid molecules into cells and assessment thereof
WO2003093469A2 (fr) * 2002-05-01 2003-11-13 Chromos Molecular Systems, Inc. Procedes d'administration de molecules d'acides nucleiques dans des cellules et evaluation associee
WO2003093469A3 (fr) * 2002-05-01 2004-04-08 Chromos Molecular Systems Inc Procedes d'administration de molecules d'acides nucleiques dans des cellules et evaluation associee

Also Published As

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EP1082419A2 (fr) 2001-03-14
AU3591299A (en) 1999-11-08
WO1999054445A3 (fr) 1999-12-09
CA2325561A1 (fr) 1999-10-28
JP2002512258A (ja) 2002-04-23
AU762986B2 (en) 2003-07-10

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