Classifications

• • 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
  • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
  • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
  • A61K38/1774—Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/19—Cytokines; Lymphokines; Interferons
  • A61K38/20—Interleukins [IL]
  • A61K38/2013—IL-2
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
  • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
• • 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
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Liposomes
  • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
  • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids

Definitions

• the invention relates to single-vial formulations of plasmid DNA/cationic lipid complexes for human clinical use, and related processes. BACKGROUND OF THE INVENTION
• plasmid DNA/cationic lipid complexes to transfer genes in vivo for the treatment of human diseases, including malignancy and cardiovascular disorders, is underway in active human clinical trials.
• HLA-B7 a foreign major histoco patibility complex protein
• Plasmid DNA was detected by polymerase chain reaction within biopsies of treated tumor nodules 3-7 days after injection but was not found in the serum at any time.
• Recombinant HLA-B7 protein was demonstrated by immunohistochemistry in tumor biopsy tissue in all five patients, and immune responses to HLA-B7 and autologous tumors could be detected. No antibodies to DNA were detected in any patient.
• One patient demonstrated regression of injected nodules on two independent treatments, which was accompanied by regression at distant sites.
• Vical Incorporated proposed a multi-center clinical trial using an improved cationic lipid mixture, DMRIE/DOPE.
• Vogelzang et al. Human Gene Therapy 5:1357 (1994); Hersh et al., Human Gene Therapy 5:1371 (1994); and Rubin et al., Human Gene Therapy 5:1385 (1994).
• the Food and Drug Administration (FDA) has authorized these clinical protocols.
• the FDA also recently allowed Vical Incorporated to conduct a clinical protocol using lipid mediated transfer of a cytokine encoding gene into tumors for treatment of malignancy. See Example 8.
• the intratumor injection of a plasmid DNA expression vector containing the human interleukin-2 (IL-2) gene reduced the incidence of tumor formation and slowed tumor growth.
• IL-2 human interleukin-2
• By local expression of cytokines at the site of the tumor it is envisioned that lower levels of cytokines will be required for efficacy as compared to systemic administration and that these levels will be sufficiently low to avoid producing toxicity in the patient.
• the findings suggest that introduction of IL-2 into a tumor, by intratumor injection of plasmid DNA expression vectors, can stimulate an antitumor response.
• plasmid DNA for therapeutic purposes requires that a pharmaceutically acceptable vehicle be found in which the DNA can be taken from the manufacturing site to the clinical site with a commercially viable interim shelf-life.
• buffers containing tris-(hydroxymethyl)aminomethane (Tris) are commonly used to handle plasmid DNA in the research laboratory, these buffers are unauthorized for parenteral use in the clinic.
• Tris tris-(hydroxymethyl)aminomethane
• a vehicle must be identified that provides for efficient handling of the plasmid DNA in the manufacturing setting, preserves the chemical and biological integrity of the plasmid DNA during shipping and storing, and allows efficient delivery of the DNA to the desired tissue target by the preferred route of administration.
• cationic lipids in a formulation with plasmid DNA introduces additional complexity into the choice of a suitable vehicle for pharmaceutical use.
• the solubility and stability of the i ⁇ dividual oppositely charged components and the complexes they form must be accommodated in a single medium. If formation and preservation of the plasmid DNA/cationic lipid complexes is considered to be essential for the functioning of the product, it is imperative that the vehicle chosen for pharmaceutical use not interfere with this interaction.
• the stability of the product can be enhanced by frozen storage, which imposes additional restrictions on the choice of vehicle for mixtures of plasmid DNA and cationic lipid.
• the stability of the complexes formed between plasmid DNA and lipid must be considered in addition to that of the individual components.
• the formulation must be designed so as to preserve the solubility and integrity of the plasmid DNA/lipid complexes over the course of storage at the frozen temperature.
• the plasmid DNA in vehicle is present in one vial.
• the lipid mixture is present in a another, separate vial either as a dried film or in solution.
• still another vial containing the diluent for rehydration of the lipid is provided to the clinic.
• the clinic staff must prepare the patient dose at the site by performing several sequential dilution and addition steps.
• the clinic staff must obtain the DNA in vehicle.
• they must obtain the lipid.
• the clinic staff may prepare dilutions of DNA/lipid complexes for the administration of escalating doses.
• the invention provides a process for making a single-vial formulation of polynucleotide/lipid complexes in a pharmaceutically acceptable vehicle for human clinical use in vivo or ex vivo comprising the steps of: (a) sterilizing a lipid solution; (b) sterilizing a polynucleotide solution; (c) combining the sterilized polynucleotide solution of step (b) with the sterilized lipid solution of step (a), in dilute form, at an ionic strength that is lower than isotonicity, to form polynucleotide/lipid complexes; and (d) adjusting the polynucleotide/lipid complexes of step (c) to near isotonicity.
• the invention further provides a process for making a single-vial formulation of plasmid DNA/cationic lipid complexes for human clinical use comprising the steps of: (a) autoclave sterilizing a cationic lipid solution at high concentration; (b) diluting the sterilized cationic lipid solution of step (a); (c) filter sterilizing a plasmid DNA solution; (d) adding the sterilized plasmid DNA solution of step (c) to the diluted sterilized cationic lipid solution of step (b) at an ionic strength that is lower than isotonicity to form DNA/lipid complexes; and (e) adjusting the DNA/lipid complexes of step (d) to near isotonicity.
• the invention also provides a process for making a single-vial formulation of plasmid DNA/cationic lipid complexes for human clinical use comprising the steps of: (a) autoclave sterilizing a cationic lipid solution at a concentration sufficiently high to substantially prevent lipid degradation during the autoclave sterilization; (b) diluting the sterilized cationic lipid solution of step (a) to a degree sufficient to substantially prevent lipid aggregation during step (d) below; (c) filter sterilizing a plasmid DNA solution; (d) adding the sterilized plasmid DNA solution of step (c) to the diluted sterilized cationic lipid solution of step (b) at an ionic strength that is lower than isotonicity to form DNA/lipid complexes; and (e) adjusting the DNA/lipid complexes of step (d) to near isotonicity.
• the invention moreover provides a process for making a single-vial formulation of plasmid DNA/cationic lipid complexes for human clinical use comprising the steps of: (a) autoclave sterilizing a cationic lipid solution having a concentration in the range of from about 0.5 to about 5.0 M; (b) diluting the sterilized cationic lipid solution of step (a) with a diluent to achieve a concentration in the range of from about 0.01 to about 1.0 M; (c) filter sterilizing a plasmid DNA solution; (d) adding the sterilized plasmid DNA solution of step (c), having a concentration in the range of from about 0.05 to about 10 mg/mL, to the diluted sterilized cationic lipid solution of step (b) at an ionic strength that is lower than isotonicity to form DNA/lipid complexes; and (e) adjusting the DNA/lipid complexes of step (d) to near isotonicity.
• the invention additionally provides a process for making a single-vial formulation of plasmid DNA/cationic lipid complexes for human clinical use comprising the steps of: (a) autoclave sterilizing a cationic lipid solution of DMRIE/DOPE, having a molar ratio in the range of from about 90:10 to about 10:90, and having a concentration in the range of from about 2 to about 10 mg DMRIE/mL; (b) diluting the sterilized cationic lipid solution of step (a) with a diluent to achieve a concentration of ⁇ _ about 2 mg OMRIE/mL; (c) filter sterilizing a plasmid DNA solution; (d) adding the sterilized plasmid DNA solution of step (c), having a concentration of _ ⁇ .
• step (d) about 10 mg plasmid DNA/mL, to the diluted sterilized cationic lipid solution of step (b) at an ionic strength that is lower than isotonicity to form DNA/lipid complexes at a mass ratio of from about 50:1 to about 1:10 DNA to OMRIE; and (e) adjusting the DNA/lipid complexes of step (d) to near isotonicity with sodium chloride.
• the invention furthermore provides a process for making a single-vial formulation of plasmid DNA/cationic lipid complexes for human clinical use comprising the steps of: (a) autoclave sterilizing a cationic lipid solution of DMRIE/DOPE, having a molar ratio of about 50:50, and having a concentration of about 8 mg DMRIE/mL; (b) diluting the sterilized cationic lipid solution of step (a) with a diluent to achieve a concentration of ⁇ __ about 1 mg DMRIE/mL; (c) filter sterilizing a plasmid DNA solution; (d) adding the sterilized plasmid DNA solution of step (c), having a concentration of _ ⁇ .
• step (d) provides a process for making a single-vial formulation of plasmid
• DNA/cationic lipid complexes in about 0.9% sodium chloride with about 1 % glycerol and about 0.01 % Vitamin E for human clinical use comprising the steps of: (a) autoclave sterilizing a cationic lipid solution of DMRIE/OOPE, having a molar ratio of about 50:50 molar ratio, and having a concentration of about 8 mg DMRIE/mL; (b) diluting the sterilized cationic lipid solution of step (a) with a diluent to achieve a concentration of ⁇ _ about 1 mg DMRIE/mL, and with glycerol and Vitamin E; (c) filter sterilizing a plasmid ONA solution;
• step (d) adding the sterilized plasmid DNA solution of step (c), having a concentration of . ⁇ _ about 5 mg plasmid DNA/mL, to the diluted sterilized cationic lipid solution of step (b) at an ionic strength that is lower than isotonicity to form DNA/lipid complexes at a mass ratio of about 5:1 DNA to DMRIE; and (e) adjusting the DNA/lipid complexes of step (d) to about 0.9% sodium chloride, wherein the final concentration of glycerol is about 1 % and of Vitamin E is about 0.01%.
• the invention provides single-vial formulations of plasmid DNA/cationic lipid complexes for human clinical use prepared by any of the above processes.
• the invention provides a single-vial formulation of plasmid DNA/cationic lipid complexes for human clinical use comprising a cationic lipid component and a plasmid ONA component, wherein the plasmid DNA component and the cationic lipid component are combined at an ionic strength that is lower than isotonicity to form the plasmid DNA/cationic lipid complexes, and further comprising a nearly isotonic aqueous medium, optionally containing no buffering agent except the plasmid DNA itself.
• the single- vial formulation may be stable in frozen, refrigerated, room temperature, or body temperature form.
• the single-vial formulation may be stable in frozen, refrigerated, or room temperature form for at least about 8 weeks.
• the single-vial formulation may further comprise about 1 % glycerol and about 0.01 % Vitamin E in nearly physiological saline.
• the invention provides a single-vial formulation for human clinical use comprising plasmid DNA/cationic lipid complexes having storage stability in frozen, refrigerated, or room temperature form for at least about 8 weeks.
• the invention provides a single-vial formulation for human clinical use comprising plasmid DNA/cationic lipid complexes retaining in vitro transfection efficacy of freshly prepared plasmid DNA/cationic lipid complexes for at least about 8 weeks.
• the objects of the invention are achieved by controlled combination of a plasmid DNA solution with a cationic lipid solution, preferably in dilute orm, at an ionic strength that is lower than isotonicity, to obtain plasmid DNA/cationic lipid complexes, which, when admixed with solutes to generate isotonicity, provide single-vial formulations suitable for human clinical use.
• in vitro transfection efficiency and prevention of lipid degradation have been produced by autoclave sterilization.
• in vitro transfection efficiency is further maximized and lipid degradation further prevented by maintaining the lipid mixtures at higher concentrations during autoclave sterilization.
• Preferred single-vial formulations of the invention have accordingly been obtained by preparing cationic lipid mixtures containing a cationic lipid constituent and a neutral lipid constituent, having a molar ratio in the range of from about 90:10 to about 10:90, preferably about 50:50.
• a cationic lipid solution is prepared from the cationic lipid mixture by hydrating a dried lipid film with a suitable diluent, preferably using water, to obtain a solution in highly concentrated form.
• the cationic lipid solution is considered to be highly concentrated by having a concentration in the range of from about 0.5 to about 5.0 M.
• This highly concentrated cationic lipid solution is subsequently subjected to sterilization by standard autoclave treatment, e.g.. 30 minutes at 121 °C, in the usual way. •7-
• a cationic lipid solution can be prepared and sterilized by filtration or irradiation or other appropriate means at any desired concentration, the higher concentration of cationic lipid solution being advantageous for protecting the material during sterilization by autoclave treatment.
• a highly concentrated cationic lipid solution Upon obtaining a highly concentrated cationic lipid solution, one preferably dilutes the solution, preferably using sterilized water (e.g., sterile WFI).
• sterilized water e.g., sterile WFI
• suitable diluents such as salines, can be substituted for water, and are selected on the basis of having low ionic strength, that is, lower than isotonicity.
• the cationic lipid solution is diluted to achieve a concentration in the range of from about 0.01 to about 1.0 M.
• suitable for ulatory agents may be introduced, as discussed below, particularly emulsifiers to facilitate the suspension of DNA and lipid during the combination step (infra). Plasmid DNA, which is pharmaceutical-grade in quality, is meanwhile obtained.
• aqueous solution such as water (e.g.. sterile WFI) or other appropriate diluent, such as saline (e.g., physiological saline), is sterilized.
• Sterilization is preferably by filtration, for instance, through a 0.2 ⁇ m membrane filter. It is preferred that the diluent have an ionic strength that is no higher than isotonicity.
• the sterilized plasmid DNA solution and diluted sterilized cationic lipid solution are usually brought to room temperature prior to combination.
• the DNA solution may be used at concentrations within a range that extends from about 0.05 to about 10 mg/mL.
• the solutions are then combined, preferably, by controlled addition of the plasmid DNA solution to the cationic lipid solution at low ionic strength with mixing to form
• DNA/lipid complexes By low ionic strength is meant lower than isotonicity. Continuous mixing of the lipid solution and plasmid DNA solution as they are combined is preferred, with a further short period of mixing after the combination is complete. The mixing may be achieved by vortexing, manual agitation (shaking), mechanical mixing or stirring, or other suitable means.
• DNA and lipid are combined to produce complexes at a mass ratio that is optimal for transfection efficiency, as evaluated, for example, by in vitro transfection assays.
• the complexes are subsequently adjusted with a to ⁇ icifier to isotonicity for physiological administration.
• a to ⁇ icifier to isotonicity for physiological administration.
• adequate sterile sodium chloride stock may be added to give a final concentration of about 0.9% NaCI (__, physiological saline).
• the final formulation can next be aseptically filled into sterile depyrogenated vials, and can then be stored frozen at about -10°C to -70°C, preferably about -20°C.
• the DNA/lipid vials are conveniently thawed and typically mixed (e.g.. by vortexing or manual agitation, _e_, shaking). They can be maintained at room temperature, and are administered advantageously within 24 hours of thawing.
• Each vial may deliver DNA in unit dosage form, or, alternatively, may constitute a multidose container.
• the single-vial formulations are provided in concentrations up to about 10 mg DNA/mL, preferably up to about 0.5 mg DNA/mL.
• an effective amount of DNA will vary with many factors, including the condition being treated, the characteristics of the -8- patient, and other factors. Typical doses will contain from about 5 mg to about 10 ⁇ g DNA, although wide variations from this range are possible while yet achieving useful results.
• physiologically acceptable excipients as called for by accepted pharmaceutical practice. These may constitute buffers, antioxida ⁇ ts, ami ⁇ o acids, emulsifiers, starches, sugars, solubilizers, surfactants, suspending agents, tonicifiers, wetting agents, etc.
• Preferred formulations are suitable for administration that is parenteral, that is, by any means other than oral.
• Parenteral administration includes injections, such as intravenous, intraarterial, intramuscular, subcutaneous, i ⁇ trader al, intraperitoneal, intratumor, and interstitial injections, infusions, and by inhalation. Injections include administration through needle/syringe and catheters.
• the invention provides single-vial formulations having final concentrations of 0.9% sodium chloride (e., physiological saline) for isotonicity, 1 % glycerol as an emulsif ier (and a cryoprotectant), and 0.01 % Vitamin E as a preservative (and an antioxidant).
• 0.9% sodium chloride e., physiological saline
• 1 % glycerol as an emulsif ier (and a cryoprotectant
• Vitamin E as a preservative (and an antioxidant).
• Cationic lipid reagents that are in use today for DNA transfection are formulated as lipid vesicles or liposomes containing cationic or positively charged lipids in combination with other lipids.
• the formulations may be prepared from a mixture of positively charged lipids, negatively charged lipids, neutral lipids, and cholesterol or a similar sterol.
• the positively charged lipid can be one of the cationic lipids, such as DMRIE, described in U.S. Patent No. 5,264,618, or one of the cationic lipids DOTMA, DOTAP, or analogues thereof, or a combination of these.
• DMRIE is 1 ,2-dimyristyioxypropy l-3-dimethyl- ydroxyethyl ammonium bromide, and is preferred. See Feigner et al., J. Biol. Chem. 269:2550 (1994).
• DMRIE can be synthesized according to Example 1.
• Neutral and negatively charged lipids can be any of the natural or synthetic phospholipids or mono-, di-, or triacylglycerols.
• the natural phospholipids may be derived from animal and plant sources, such as phosphatidylcholine, phosphatidylethanolamine, sphingo yelin, phosphatidylserine, or phosphatidylinositol.
• Synthetic phospholipids may be those having identical fatty acid groups, including, but not limited to, dimyristoylphosphatidylcholine, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine and the corresponding synthetic phosphatidylethanolamines and phosphatidylglycerols.
• the neutral lipid can be phosphatidylcholine, cardiolipin, phosphatidylethanolamine, mono-, di- or triacylglycerols, or analogues thereof, such as dioleoylphosphatidylethanolamine (DOPE), which is preferred.
• DOPE can be purchased from Avanti Polar Lipids (Alabaster, Ala).
• the negatively charged lipid can be phosphatidylglycerol, phosphatidic acid or a similar phospholipid analog.
• lipids such as cholesterol, glycolipids, fatty acids, sphi ⁇ golipids, prostaglandins, gangliosides, neobee, nioso es, or any other natural or synthetic amphophiles can also be used in liposo e formulations, as is conventionally known for the preparation of liposomes.
• the cationic lipid in a composition for preparing cationic liposomes, can be present at a concentration of between about 0.1 mole % and 100 mole %, preferably 5 to 100 mole %, and most preferably between 20 and 100 mole %
• the neutral lipid can be present in a concentration of between about 0 and 99.9 mole
• the negatively charged lipid can be present at between about 0 to 49 mole % and preferably 0 to 40 mole %.
• Cholesterol or a similar sterol can be present at 0 to 80 mole %, and preferably 0 to 50 mole %.
• Lipid compositions having at least one amphipathic lipid can spontaneously assemble to form liposomes.
• Lipid reagents having a cationic lipid species can be prepared as cationic liposomes.
• the component lipids can be dissolved in a solvent such as chloroform.
• the mixture can be evaporated to dryness as a film on the inner surface of a glass vial.
• the amphipathic lipid molecules On suspension in an aqueous solvent, the amphipathic lipid molecules will assemble themselves into liposomes. See Example 2.
• the liposomes can be analyzed for potency by in vitro transfection assays, in these assays, plasmid DNA and cationic lipid complexes are formed by mixing of the two separately diluted components. The mixture is then added to cells in culture and transfection assessed according to the procedure of Example 3.
• PLASMID DNAs PLASMID DNAs
• These plasmids accordingly can be selected from among prokaryotic and eukaryotic vectors, pBR322- and pUC-based vectors, and their derivatives, etc. They can utilize any of various origins of replication, for instance, prokaryotic origins of replication, such as pMB1 and ColEI, and eukaryotic origins of replication, such as those facilitating replication in yeast, fungi, insect, and mammalian cells (e.g.. SV40 ori).
• prokaryotic origins of replication such as pMB1 and ColEI
• eukaryotic origins of replication such as those facilitating replication in yeast, fungi, insect, and mammalian cells (e.g.. SV40 ori).
• genes can incorporate any of numerous genetic elements to facilitate cloning and expression, such as selectable genes, polylinkers, promoters, enhancers, leader peptide sequences, i ⁇ trons, translation facilitators, Kozak sequences, polyadenylatio ⁇ signals, transcription terminators, 5' UTRs, 3' UTRs, etc.
• selectable genes such as selectable genes, polylinkers, promoters, enhancers, leader peptide sequences, i ⁇ trons, translation facilitators, Kozak sequences, polyadenylatio ⁇ signals, transcription terminators, 5' UTRs, 3' UTRs, etc.
• the selection of vectors, origins, and genetic elements will vary based on requirements and is well within the skill of workers in this art.
• Genes encoding any of diverse structural proteins can be inserted into the plasmids for delivery into cells.
• These genes may constitute genomic DNA, cDNA, synthetic DNA, polynucleotide, oligonucleotide, etc. sequences. Transfer of mRNA, a ⁇ tisense oligomers, and triple helix agents are also expressly contemplated as falling within the scope of the present invention.
• sequences may be obtained using chemical synthesis or gene manipulation techniques. They can be inserted into plasmids, and the plasmids subsequently introduced into host cells for propagation.
• Host cells can be selected from among prokaryotes and eukaryotes, including bacterial, yeast, fungi, insect and mammalian cells.
• Preferred hosts are bacteria, such as E. coli. Any suitable strain of E. coli is contemplated.
• Propagation of plasmid DNA-containi ⁇ g hosts can be carried out using known processes. Such processes may utilize incubators, bioreactors, fermentors, etc., according to batch fermentation, fed batch fermentation, continuous culture, Type I, II, and III fermentation, aseptic fermentation, consortium fermentation, protected fermentation, etc. Fitting the conditions (e.g.. medium, temperature, pH, hours, agitation, aeration, etc.) for propagation to the circumstances is empirical and well within the skill of those in the art.
• conditions e.g. medium, temperature, pH, hours, agitation, aeration, etc.
• plasmid DNA Purification of plasmid DNA to pharmaceutical grade quality may proceed using well established processes. Alternatively, processes for production of pharmaceutical grade plasmid DNA disclosed in Example 8 are preferred. Additionally, processes for reducing RNA concentrations in cell lysates using diatomaceous earth materials also according to Example 8 are also preferred in purifying plasmid DNA to pharmaceutical grade standards.
• DMRIE/DOPE cationic lipid mixtures are prepared at from about 90:10 to about 10:90 molar ratio, preferably at about 50:50 molar ratio.
• a cationic lipid solution is prepared from the cationic lipid mixture by hydrati ⁇ g a dried lipid film with a suitable diluent, preferably with water, at >_ about 2 mg DMRIE/mL, preferably at about 2 to about 10 mg DMRIE/mL, most preferably at about 8 mg DMRIE/mL.
• This highly concentrated cationic lipid solution is subsequently subjected to sterilization by standard autoclave treatment, e , 30 minutes at 121 °C, in the usual way.
• DMRIE/DOPE lipid solution Upon obtaining an autoclaved highly concentrated DMRIE/DOPE lipid solution, one then dilutes the solution, preferably with sterilized water ⁇ ___, sterile WFI), to ⁇ _ about 2 mg DMRIE/mL, preferably to _ ⁇ _ about 1 mg DMRIE/mL, most preferably to ⁇ _ 0.2 mg DMRIE/mL.
• suitable diluents such as salines, can be substituted for water, and are selected on the basis of having low ionic strength, that is, lower than isotonicity.
• suitable formulatory agents may be introduced, advantageously by addition into the diluent, for subsequent admixture into the cationic lipid solution.
• emulsifiers may be introduced, preferably glycerol, in amounts to provide a final concentration in the single-vial formulation of from about 0.5 to about 5% glycerol, preferably 1 % glycerol.
• preservatives may also be added at this time, preferably Vitamin E, in amounts to give a final concentration in the single-vial formulation of from about 0.005 to about 0.05% Vitamin E, preferably about 0.01 % Vitamin E.
• Plasmid DNA that has been purified to pharmaceutical-grade quality is meanwhile obtained.
• a process for the production of pharmaceutical-grade plasmid DNA is provided in Example 4.
• the plasmid DNA in aqueous solution, such as water (e.g.. sterile WFI) or other appropriate diluent, such as saline (e.g., physiological saline), is sterilized.
• Sterilization is preferably by filtration, for instance, through a 0.2 ⁇ m membrane filter. It is preferred that the diluent have an ionic strength that is no higher than isotonicity.
• the sterilized plasmid DNA solution and diluted sterilized DMRIE/DOPE lipid solution are usually brought to room temperature prior to combination.
• the DNA may be used at concentrations within a range that extends from concentrations that are quite high, for example, 10 mg/mL, to concentrations that are quite low, for example, 0.02 mg/ml.
• the solutions are then combined, preferably, by controlled addition of the plasmid DNA solution to the DMRIE/DOPE solution with mixing to form DNA/lipid complexes at low ionic strength. Low ionic strength means lower than isotonicity.
• DNA and lipid are combined to produce complexes at a mass ratio of from about 50:1 to about 1:10
• DNA to DMRIE preferably at a mass ratio of from about 10:1 to about 1 :5 DNA to DMRIE, most preferably at a mass ratio of about 5:1 DNA to DMRIE.
• the complexes are adjusted with solutes to isotonicity for physiological administration.
• the invention provides single-vial formulations having final concentrations of 0.9% sodium chloride, 1 % glycerol, and 0.01 % Vitamin E.
• plasmids containing reporter genes for instance, iuciferase and ⁇ -galactosidase
• plasmids operatively encoding polypeptides suitable for human gene therapy ejj., HLA-B7 and human IL-2. See Examples 5-10.
• DMRIE cationic lipid species and cationic lipid mixtures
• DOSPA DOSPA/DOPE
• HP-DORIE HP* DORIE/DOPE
• r- U-DMRIE r-MU-DMRIE/DOPE
• r-MC-DMRIE r MC DMRIE/DOPE
• rf-Ser-DMRIE (5-Ser- DMRIE/DOPE, ⁇ AE-DMRIE, ⁇ AE-DMRIE/D0PE
• Arabi ⁇ ose-TU-rDMRIE,Arabi ⁇ ose-TU-rDMRIE/DOPE Galactose- TU-r-DMRIE, Galactose TU r DMRIE/DOPE, Glucose-TU-r-DMRIE, and Glucose TU r DMRIE/DOPE.
• Single-vial formulations of the invention containing DNA/DMRIE-DOPE at a mass ratio of 5:1 DNA to cationic lipid in 0.9% sodium chloride with 1% glycerol and 0.01 % Vitamin E demonstrated no signs of toxicity in acute intravenous toxicity studies in mice, repeat dose safety studies in mice, and repeat dose safety studies in cyno olgus monkeys.
• the single-vial formulations of the invention are suitable for human clinical use in vivo (Example 12- 13) or ex vivo (see U.S. Patent No. 5,399,346 to Anderson et al. for "Gene Therapy”).
• DMRIE was synthesized according to Example 1. DOPE was purchased from Avanti Polar Lipids
• Cationic liposomes were prepared by mixing a chloroform solution of the lipids in a sterile glass flask. The solvent was removed by evaporation under reduced pressure to produce a dried lipid film. Vials were placed under vacuum overnight to remove any solvent traces. The lipid mixture was hydrated by addition of sterile water for injection.
• EXAMPLE 3 IN VITRO TRANSFECTION PROTOCOLS Plasmid DNA/cationic lipid complexes were prepared by mixing an aliquot of an polynucleotide solution with an aliquot of a iiposome solution at room temperature. Different ratios of positively charged liposomes to polynucleotides can be used to suit the need. The methods are a modification of those described in Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413 (1987), and Feigner and Holm, Focus 11(2) Spring, 1989.
• Transfections were carried out in 96-well plates, as follows: (1) The wells of a 96-well microtiter plate were seeded with 20,000 to 40,000 cells per well; (2) Single-vial formulations of plasmid DNA/cationic lipid complexes were prepared in sterile vials or tubes;
• the cells were washed with an additional volume of cell culture medium without fetal calf serum, and the wash medium was removed by aspiration; (6) A volume of the diluted plasmid DNA/cationic lipid complexes was added to the washed cells in a well of the microtiter plate; the volume transferred usually consisted of 80 to 100 ⁇ L;
• /ff-galactosidase was the reporter gene
• the expression was monitored colorimetrically, using chlorophenyl red- ?-galactopyranoside (CPRG) as a substrate, reading the plates with a microtiter reader at
• Solution I 61 M glucose + 25 M Tris buffer pH 8.0 + 10 mM EDTA at 5°C
• 12 mL per gram wet bacterial weight Solution II 0.2 N NaOH + 1% SDS
• 9 mL per gram wet bacterial weight of cold Solution III 3.0 M potassium acetate pH 5.0 at 5°C
• the supernatant was collected and clarified by adding approximately 25 g/1 Celite ® diatomaceous earth and filtering through a (preferably precoated) filter membrane (Whatman If 1, 113 or equivalent) arranged in a table top Buchner funnel.
• a filter membrane Whatman If 1, 113 or equivalent
• the cell debris was removed from the lysate by direct Celite ® aided filtration.
• approximately 90 g/l Celite ® diatomaceous earth was added directly to the lysis solution and mixed by swirling until homogenous.
• the lysate was then filtered through a (preferably precoated) filter membrane (Whatman ⁇ 1, 113 or equivalent) arranged in a table top Buch ⁇ er funnel.
• RNA, Protein and Lipopolysaccharide Removal Ammonium acetate was added to the TE buffer to 2.5 M and stirred for approximately 30 minutes at 2-8°C.
• the suspension, which still contained diatomaceous earth, was filtered through a (preferably precoated) filter membrane arranged in a table top Buchner funnel.
• the DNA filtrate was then optionally clarified by sub-micron filtration.
• the S-1000 matrix was an inert and highly stable matrix that was prepared by covalently cross-linking ally) dextran with N,N'-methylenebisacrylamide.
• the column was poured in two Pharmacia XK26/100 columns (Pharmacia, Piscataway, NJ) with a final bed height of 80-85 cm (2.6x80cm) resulting in a total column volume of approximately 900 mL and a total length of approximately 160 cm.
• the columns were individually pressure packed in one direction, reversed and connected in series for equilibration and operation.
• HLA-B7 encoding plasmid was constructed of about 5000 bp in size. It derived from a pBR322- based plasmid containing a bacterial origin of replication. It encoded the heavy (human HLA-B7 cDNA) and light (chimpanzee ⁇ -2 microglobulin cDNA) chains of a Class 1 MHC antigen designated HLA-B7. These two proteins were expressed on a bi-cistronic mRNA. Eukaryotic cell expression of this mRNA was dependent on a Rous Sarcoma Virus (RSV) promoter sequence derived from the 3' Long Terminal Repeat (LTR). Expression was also dependent on a transcription termination/polyadenylation signal sequence derived from the bovine growth hormone gene.
• RSV Rous Sarcoma Virus
• LTR 3' Long Terminal Repeat
• Expression of the heavy chain was regulated by the 5' cap-dependent protein translation start site.
• Expression of the light chain was regulated by a Cap Independent Translational Enhancer (CITE) sequence derived from the Encephalomyocarditis Virus.
• CITE Cap Independent Translational Enhancer
• the plasmid also encoded a kana yci ⁇ resistance gene derived from Tn903.
• Cells were a > 75% confluent monolayer prior to transfection.
• the cells were transfected with 10 ⁇ g plasmid DNA in the presence of 2 ⁇ g DMRIE (synthesized in house) and 2 ⁇ g DOPE (purchased from Avanti Polar Lipids, Alabaster, Ala).
• the cells were incubated at 37°C, 5% C0 2 throughout.
• Reduced serum media, ___, Opti-MEM® reduced-serum media (GIBC0 BRL Life Technologies, Gaithersburg, MD), supplemented with fetal calf serum, was added to the cells 1-4 hours and 24 hours post-transfection. Cells were harvested 48 hours post-transfection.
• HLA-B7 expression on the cell surface was measured by labelling with anti-HLA-B7 mouse antibody, followed by a fluorescent secondary antibody (anti-mouse IgG monoclonal antibody R-phycoerythrin conjugate). Immu ⁇ ofluorescent staining of the cells was analyzed by flow cytometry. A two-fold increase in mean fluorescence intensity was observed for transfected cells in contrast to negative controls (non-transfected cells or cells transfected with an irrelevant gene). Potency was equivalent to that of freshly prepared plasmid DNA/cationic lipid complexes.
• IL-2 plasmid DNA/DMRIE-DOPE lipid complexes intended for use in human gene therapy, demonstrated in vitro potency as single-vial formulations of the invention.
• a plasmid encoding IL-2 was constructed of about 5000 bp in size. It derived from a pUC-based plasmid containing a bacterial origin of replication. It encoded an IL-2 fusion protein. The protein was constructed by cloning a portion encoding a short segment of the 5' untranslated region and the first six amino acids of the leader peptide of the rat insulin II gene 5' of the human IL-2 coding sequence minus the first two amino acids of its leader peptide. This fusion protein was placed under the eukaryotic tra ⁇ scriptional control of the cyto episcopovirus (CMV) immediate early 1 promoter/enhancer sequence.
• CMV cyto egalovirus
• This sequence facilitated expression of a composite mRNA containing a 5' untranslated sequence from the CMV immediate early 1 gene, including the 800-*- bp intron, the IL-2 fusion protein coding sequence, and a 3' untranslated sequence derived from the bovine growth hormone gene having transcription termination/polyadenylation signal sequence.
• the plasmid also encoded a kanamyci ⁇ resistance gene derived from Tn903.
• EXAMPLE 9 PHARMACEUTICAL-GRADE PURIFIED IL-2 PLASMID DNA The IL-2 encoding plasmid was purified to pharmaceutical-grade standards as determined by the criteria given in Table 2 below.
• B16F0 cells From 200,00 to 400,000 B16F0 cells were seeded per well into a 6-well plate the day before transfection. Cells were a > 75% confluent monolayer prior to transfection. The cells were transfected with 2.5 ⁇ g plasmid DNA in the presence of 0.5 ⁇ g DMRIE (synthesized in house) and 0.5 ⁇ g DOPE (purchased from Avanti Polar Lipids, Alabaster, Ala). The cells were incubated at 37°C, 5% C0 2 throughout.
• a reduced serum medium, ej Opti-MEM® reduced-serum media (GIBCO BRL Life Technologies, Gaithersburg, MD), supplemented with fetal calf serum, was added to the cells at commencement of transfection and 24 hours post-transfection. Cell supernatant was harvested 48 to 80 hours post-transfection. IL-2 expression in the cell supernatant was measured by an enzyme amplified sensitivity immunoassay (Medgenix ELISA, Medgenix Diagnostics, Fleurus, Belgium). Potency was equivalent to that of freshly prepared plasmid DNA/cationic lipid complexes.
• Vitamin E retained full stability over 57 days of storage at -20°C and 2°C.
• IL-2/DMRIE-D0PE complexes retained apparent full activity over 57 days of storage; free DNA showed a half- life of 500 days for conversion from circular to linear form at this temperature.
• IL-2/DMRIE-D0PE complexes showed a half-life of 34 days; free DNA was converted from circular to linear form with a 290 day half-life at this temperature.
• Vitamin E In comparison, in the absence of Vitamin E, IL-2/DMRIE-D0PE complexes showed a half-life of 12 days at 37°C; and free DNA was converted from circular to linear form with a 108 day half -life at this temperature. Therefore, the presence of Vitamin E at the level of 0.01 % in the subject embodiment provided 2.7 to 3 times greater stability to the components of the vial.
• the batch amount of DMRIE Br was weighed out.
• the neck of the round bottom flask was rinsed with about five (5) mL of chloroform and the contents swirled gently for at least one minute to mix. Any solution adhering to the neck after swirling was rinsed into the flask with additional chloroform. Using a rotary evaporator, the chloroform was removed from the solution from above. The flask was kept on the rotary evaporator until condensate was no longer visible in the condenser.
• a desiccator was thoroughly wiped down with alcohol and placed in a ventilated laminar flow hood. The round bottom flask from above was placed in the desiccator. The desiccator was connected to a vacuum pump, the desiccator evacuated, and the round bottom flask maintained under vacuum for at least
• the desiccator was isolated from the vacuum pump.
• the desiccator was connected to a nitrogen gas source, the gas turned on, and the vacuum released.
• the gas source was removed.
• the round bottom flask was removed from the desiccator. Using a sterile disposable pipet, sterile water for injection was added to the flask. The flask was capped, the liquid swirled, and vortexed for at least 5 minutes or until rehydration was achieved.
• Each vial was capped with a clean teflon-coated gray butyl stopper, and the cap secured with an aluminum crimp.
• the filled vials from above were autoclaved using a standard liquid cycle on the autoclave (no less than 121 °C for 30 minutes). If autoclaved vials were not used immediately, they were stored at 2 to 8°C. The autoclaved vials were not held at room temperature for more than 6 hours.
• the plasmid DNA standard bulk solution was filtered through a 0.2 ⁇ m sterile filtration unit.
• the filtrate was collected in a sterile disposable tube.
• the tube was stored at 15 to 30°C.
• sterile water for injection was transferred into a sterile disposable 250-mL bottle.
• An appropriate amount of stock glycerin and an appropriate amount of stock Vitamin E was added to the bottle from above and swirled to mix.
• IL 2/DMRIE-D0PE COMPLEXES IN PHASE I HUMAN CLINICAL TRIALS Direct intratumor injection of plasmid DNA expression vectors provides a method for the introduction of IL-2 genes into the tumor.
• the sponsor tests for safety and dose optimization of the direct gene transfer approach for delivering the IL-2 gene directly into solid tumors and lymphomas. Expression of the IL-2 gene is confirmed.
• the IL-2 produced should elicit an immunologic antitumor response which in turn may lead to a systemic immunological elimination of other tumor cells. Dose responses are correlated with specific immune responses.
• the phase I protocol is designed: 1) to minimize the risks to the patient;
• the objectives of the clinical plan include:
• the product is composed of plasmid DNA coding for IL-2, formulated in an injection vehicle with the cationic lipid mixture DMRIE/DOPE.
• the lipid When introduced into the target tumor tissue, the lipid facilitates transfection of cells with the plasmid.
• the recombinant gene is expressed.
• the lipid mixture is a combination of two compounds: DOPE (CAS name: 1,2-dioleoyl-sn-glycero- 3-phosphoethanolamine) and DMRIE synthesized as DMRIE-Br (CAS name: (+/-)-N-(2-hydroxyethyl)-N,N- dimethyl-2,3-bis(tetradexyloxy)-1-propanaminium bromide) which are assumed to be rapidly metabolized.
• DOPE CAS name: 1,2-dioleoyl-sn-glycero- 3-phosphoethanolamine
• DMRIE-Br CAS name: (+/-)-N-(2-hydroxyethyl)-N,N- dimethyl-2,3-bis(tetradexyloxy)-1-propanaminium bromide
• the plasmid/lipid mixture and injection vehicle are produced in accordance with the methodology of the invention.
• the plasmid DNA is formulated with DMRIE/DOPE lipid mixture in the injection vehicle which constitutes 1 % glycerol and 0.01% Vitamin E in normal aqueous saline (0.9% sodium chloride in sterile water for injection).
• the dosage form is delivered by injection into solid tumor tissue.
• the concentration of plasmid DNA and DMRIE/DOPE in each dose package is specified in the following table:
• Eligible patients have a primary tumor nodule injected several times at specified intervals with a specified dose of the study drug (see below). There are four groups with 5 patients each treated at the prescribed dose (10, 30, 100 or 300 ⁇ g), with a group of 5 patients retreated at the maximum tolerated dose (MTD), or at 300 ⁇ g if the MTO is not reached. The highest dose that does not yield Grade 3 or higher toxicities is considered the MTD. All toxicities are graded according to the World Health Organization
• the study drug is administered and toxicities are monitored.
• Tumor lesions are selected for treatment if they are accessible to intratumor administration by direct needle injection. These metastatic lesions are located at any accessible site such as skin, nodes, lung, liver, soft tissues etc. Bony tumors are excluded.
• the amount of study drug material injected into each tumor is based on the algorithm outlined below.
• the prescribed dose (10, 30, 100 or 300 ⁇ g) is thawed and diluted with injection vehicle to the appropriate volume. If necessary, the study drug is injected with the aid of sonographic or CAT scan visualization of the metastasis.
• gentle aspiration is applied to the syringe to ensure that no material is injected intravenously. After injection of the drug and with the needle still in place, the dead space is flushed with 0.25-0.50 mL of sterile normal saline (0.9% sodium chloride in sterile water for injection).
• Vital signs are measured every 15 minutes at the start of, during, and after the injection for at least 2 hours or until the patient is stable. If the systolic blood pressure drops below 80 mm Hg, the injection is terminated immediately and the patient is closely monitored and treated appropriately until blood pressure is normalized.
• tumor sizing is done at each intratumoral injection of the nodule. If the tumor shrinks to a point where it can no longer be injected, subsequent doses are administered into another tumor nodule if any are present.
• patient follow-up includes evaluations with tumor sizings at weeks 8 and 16. After the week 16 visit, patients are evaluated a minimum of every 4 months.
• IL-2 The following parameters are measured to evaluate the tumor transfection and expression of IL-2: 1) the presence of DNA from the IL-2 gene is assessed by PCR amplification of cells obtained by biopsy of the treated site after the injection of the study drug, 2) immunohistochemical staining of tumor biopsy samples is used to assess immu ⁇ ologic response and soluble IL-2 expression, 3) serum IL-2 levels are measured pre-treatment and 2 times post the start of therapy, however, the detection of serum IL-2 levels is not anticipated due to IL-2 instability, 4) PCR analysis of peripheral blood samples is used to test for the presence of plasmid DNA after the start of treatment and compared to pre-therapy, but detection of the gene in peripheral blood samples is not anticipated, 5) the cellular immune response is evaluated by measuring baseline and post-treatment IL-2 induced activation of PBMC by thymidine uptake assay and NK/ LAK response in peripheral blood pre-therapy and post-therapy, and 6) an attempt is made to excise tumor tissue from another site prior to treatment for diagnosis. immuno
• the clinical response is measured. Standard oncologic criteria are applied to determine whether or not a patient responds to the study drug. All tumor measurements are recorded in centimeters and constitute the longest diameter and the perpendicular diameter at the widest portion of the tumor. The tumor response definitions listed below are used to compare current total tumor size to pre-treatment total tumor size.

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• 1996
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