WO1999059638A2 - Compositions and methods for the delivery of nucleic acid molecules - Google Patents
Compositions and methods for the delivery of nucleic acid molecules Download PDFInfo
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- WO1999059638A2 WO1999059638A2 PCT/US1999/010697 US9910697W WO9959638A2 WO 1999059638 A2 WO1999059638 A2 WO 1999059638A2 US 9910697 W US9910697 W US 9910697W WO 9959638 A2 WO9959638 A2 WO 9959638A2
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- 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
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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- 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
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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 generally to therapeutic compositions and methods, and more specifically, to compositions and methods which are useful for facilitating the uptake of nucleic acids by animal cells.
- nucleic acids Since the discovery of nucleic acids in the 1940s and continuing through the most recent era of biotechnology, substantial research has been undertaken in order to affect the course of a disease through interaction with the nucleic acids of living organisms. Most recently, a wide variety of methods have been described for altering or affecting genes within humans or animals, by directly administering to the human or animal a nucleic acid molecule which alters or effects the course of a disease.
- vectors have been utilized to deliver nucleic acid molecules to a human or animal, including for example, viral vectors derived from retroviruses, adenoviruses, vaccinia viruses, herpes viruses, and adeno-associated viruses (see Jolly, Cancer Gene Therapy 7(l):51-64, 1994), as well as direct transfer techniques such as lipofection (Feigner et al., Proc. Natl. Acad. Sci.
- Intramuscular injection of pDNA in saline results in a very small amount of injected pDNA being taken up by cells and expressed, while the majority of the pDNA is rapidly degraded or removed from the muscle (Man horpe et al., Human Gene Therapy, 1993, 4, 419-431).
- viscosity-increasing polymers such as polyvinylpyrrolidone and polyvinylalcohol (Rolland, A. and Mumper, R.J., WO 96/21470).
- the inclusion of such polymers in large amounts needed to induce favorable responses may be limited by: ease of administration, variability in precise amounts available in the microenvironment to induce biological responses.
- the present invention discloses novel compositions and methods for facilitating nucleic acid uptake in cells either in vivo or ex vivo, and further provides other related advantages.
- compositions for delivering nucleic acid molecules to an animal cell comprising a recombinant or synthetic nucleic acid molecule in an aqueous solution comprising 0.5% to 5% (w/v) neutral or negatively charged polysaccharide, or in a lyophilized composition comprising a neutral or negatively charged polysaccharide.
- the neutral or negatively charged polysaccharide may be limited to a selected molecular weight (e.g., an average molecular weight of between about 1,000 and about 3,000, 4,000, 5,000, 6,000, 7,000, or, 8,000 daltons one preferred range is between 1,500 and 3,500 daltons), or to a particular species or combination of two or more species of polysaccharides (e.g., mannan, dextran (such as maltodextin), hyaluronic acid, or a glycosaminoglycan such as heparan sulfate, chondroitin sulfate or dermatan).
- mannan such as maltodextin
- hyaluronic acid such as heparan sulfate, chondroitin sulfate or dermatan
- a glycosaminoglycan such as heparan sulfate, chondroitin sulfate or dermatan.
- the compositions provided herein may
- compositions and methods are provided for delivering nucleic acid molecules to an animal cell.
- compositions comprising a recombinant or synthetic nucleic acid molecule in an aqueous solution comprising 0.5% to 5% (w/v) polyalkylene glycol, or in a lyophilized composition comprising polyalkylene glycol.
- the polyalkylene glycol may be limited to a selected molecular weight (e.g., an average molecular weight of between about 1,000 and about 3,000, 4,000, 5,000, 6,000, 7,000, or, 8,000 daltons), or to a particular species or combination of species of polyalkylene glycols (e.g., polypropylene glycol, polyethylene glycol, or, methoxy-polyethylene glycol).
- the compositions provided herein may contain further ingredients, such as for example, a mannan.
- compositions may be utilized to deliver a nucleic acid molecule to an animal cell.
- Particularly preferred methods for delivering the nucleic acid molecule include intravenous, intramuscular, intraocular, intrapericardial, subcutaneous, intrarticular, intrathecal and mucosal modes of administration.
- the compositions provided herein may be directly administered to a tumor, for example, by stereotatic injection.
- a wide variety of nucleic acid molecules may be prepared and/or utilized in the compositions and methods provided herein.
- the nucleic acid molecule may be a vector that directs the expression of a protein or polypeptide, or which contains an antisense molecule or a ribozyme sequence.
- pro- drug converting enzymes such as thymidine kinase, or, cytosine deaminase
- therapeutic molecules such as, for example, thrombopoietin ("TPO"), erythropoietin, Factor VIII, Factor IX, insulin, growth hormones, leptin and antibodies.
- the protein or polypeptide may encode an antigen (e.g., from a foreign organism such as a virus, bacteria or parasite, or from a pathogenic cell such as an autoreactive immune cell or a cancer cell), and or, an immunomodulatory co-factor such as IL-2, IL-3, IL-4, IL-10, IL-12, IL-15, or, gamma interferon.
- the vector may direct the expression of at least two different recombinant or synthetic nucleic acid molecules (e.g.. an immunomodulatory co-factor and a prodrug converting enzyme, or a therapeutic protein or polypeptide such as erythropoietin and a prodrug converting enzyme).
- Figure 1 is a bar graph which shows reporter gene expression in mouse TA muscle after intramuscular injection of DNA formulations including 2% hyaluronic acid, 2% maltodextrin, 2% PEG (2000). and a saline control (naked DNA).
- Figure 2 is a bar graph which shows luciferase reporter gene expression after intramuscular injection of DNA formulations including 2% maltodextrin, and saline (naked DNA control). Data is from pooled results collected using a total of 93 muscles.
- Figure 3 is a bar graph which shows CAT activity in mice after intramuscular injection of DNA formulations including 2% maltodextrin, and saline (naked DNA control). Data is from pooled results collected using a total of 20 muscles.
- Figure 4 is a bar graph which shows the ELISA results of expression of recombinant mouse erythropoietin in vivo by administration using maltodextrin at 75 and 150 ug as compared to DNA saline controls, after one and two weeks.
- Figure 5 is a schematic illustration of luciferase plasmid CMVKM L.UC.
- Figure 6 is a graph which shows viscosity measurements of placebo formulations containing varying amounts of maltodextrin.
- Figure 7 is a bar graph that shows serum erythropoietin levels after intramuscular injection of EPO DNA formulations in mice.
- Figure 8 is a graph that shows the percent hematocrit after intramuscular injection of various amounts of EPO DNA formulations in mice.
- Figure 9(a) shows serum EPO levels after intramuscular injection of EPO DNA formulations in SCID mice.
- Figure 9(b) shows the percent hematocrit of the mice.
- Nucleic acid molecule refers to any deoxyribonucleic acid (DNA), ribonucleic acid (RNA), oligonucleotide, or nucleic acid fragment generated by polymerase chain reaction, ligation, scission, endonuclease action, or, exonuclease reaction.
- Nucleic acid molecules can be composed of monomers that are naturally- occurring (e.g., DNA or RNA), or modified monomers (e.g., modifications to sugar portions and/or pyrimidine or purine portions of a nucleic acid).
- nucleic acids may be linked by phosphodiester bonds, or suitable analogs such as phosphorothioate, phosphorodithioate, phosphoramidate, and the like.
- Nucleic acid molecules may be single-stranded, double-stranded, or chimeric single- or double- stranded molecules. Nucleic acid molecules may be of any desired length, including for example, as small as 8 to 10 nucleotides, or as large as an entire gene.
- isolated nucleic acid molecule refers to a nucleic acid molecule that is not integrated into the genomic DNA of an organism. Examples include both recombinantly and synthetically generated nucleic acid molecules.
- a "promoter” is a nucleic acid sequence that directs the transcription of a structural gene. Typically, a promoter is located in the 5' region of a gene and is proximal to the transcriptional start site of the structural gene.
- Nucleic acid expression vector refers to an assembly which is capable of directing the expression of a sequence or gene of interest.
- the vector must include a promoter which, when transcribed, is operably linked to the sequence(s) or gene(s) of interest, as well as a polyadenylation sequence.
- the nucleic acid expression vectors described herein may be contained within a plasmid construct.
- the plasmid construct may also include a bacterial origin of replication, one or more selectable markers, a signal which allows the plasmid construct to exist as single-stranded DNA (e.g., a M13 origin of replication), a multiple cloning site, and a "mammalian" origin of replication (e.g., a SV40 or adenovirus origin of replication).
- the vector may be comprised one or more viral nucleic acids, e.g., a viral promoter and/or envelope sequence.
- Polyalkylene glycols refer to 2 or 3 carbon polymers of glycols.
- Two carbon polyalkylenes include polyethylene glycol (PEG) of various molecular weights, and its derivatives, such as polysorbate, polyoxyethylene sorbitan monolaurate, and polyethylene glycol- -isooctylphenyl ether.
- Three carbon polyalkylenes include polypropylene glycol and its derivatives.
- Polysaccharides refer to polymers of monomer sugars. These sugars include D-glucose, D-mannose, D-fructose, D-galactose, L-galactose, D-xylose and D- arabinose.
- derivative monosaccharides may also be polymerized. Such derivatives include D-glucuronic acid, N-acetyl-muramic acid, and N-acetyl neuraminic acid. Polymers of these monosaccharides may be composed of one type of saccharide or multiple types of saccharides and may be various molecular weights.
- Naturally occurring polysaccharides that may be used within the scope of this invention include dextrans of various molecular weights (including maltodextrin), a-amylose, amylopectin, amylase-modified versions of polysaccharides, fructans, mannans, xylans, and arabinans.
- Immunogenicity refers to the ability of a given molecule or a determinant thereof to induce the generation of antibodies upon administration in vivo, to induce a cytotoxic response, activate the complement system, allergic reactions, and the like.
- An immune response may be measured by assays that determine the level of specific antibodies in serum, by assays that determine the presence of a serum component that inactivates the condensing agent/nucleic acid complex or conjugated gene delivery vehicle, or by other assays that measure a specific component or activity of the immune system. Low immunogenicity may be established by these assays.
- the terms "low immunogenicity,” “reduced immunogenicity,” “lowered immunogenicity” or similar terms are intended to be equivalent terms.
- compositions and methods of delivery of nucleic acids to cells in vivo comprise a recombinant or synthetic nucleic acid molecule in an aqueous solution comprising 0.5% to 5% (w/v) polyalkylene glycol, and/or a 0.5% to 5% (w/v) neutral or negatively charged polysaccharide.
- the compositions can be provided as a lyophilized composition comprising a polyalkylene glycol or a neutral or negatively charged polysaccharide.
- compositions provide a greatly increased level of expression of a desired nucleic acid molecule, as compared to compositions wherein the nucleic acid molecule is delivered in saline alone.
- the compositions provide a high local concentration of other nucleic acids such as, oligonucleotides, ribozymes, etc., then taken up by cells, they may exert their enhanced biological activities.
- Nucleic acid molecules that can be generated for use within the compositions described herein include both coding and noncoding nucieic acid molecules.
- suitable molecules include, for example, cytotoxic genes, disease-associated antigens, antisense sequences and ribozyme molecules, sequences which encode gene products that activate a compound with little or no cytotoxicity (i.e., a "prodrug") into a toxic product, sequences which encode immunogenic portions of disease-associated antigens, replacement genes and sequences which encode immunomodulatory cofactors or immune accessory molecules.
- cytotoxic genes include the genes which encode proteins such as ricin (Lamb et al., Eur. J. Biochem.
- Antisense molecules are those nucleic acid molecules which are capable of forming a stable duplex or triplex with a mRNA transcript or gene of interest. Antisense molecules can be utilized not only to inhibit or prevent transcription or translation of a desired gene or transcript, but also as a cytotoxic gene in order to induce a potent Class I restricted response. Briefly, in addition to binding RNA and thereby preventing translation of a specific mRNA, high levels of specific antisense sequences may be utilized to induce the increased expression of interferons (including gamma- interferon), due to the formation of large quantities of double-stranded RNA. The increased expression of gamma interferon, in turn, boosts the expression of MHC Class I antigens. Preferred antisense sequences for use in this regard include actin RNA, myosin RNA, and histone RNA.
- Ribozyme nucleic acid molecules are those molecules which contain a catalytic center. Ribozymes include not only RNA enzymes, but self-splicing RNAs, self-cleaving RNAs, and nucleic acid molecules that perform these catalytic functions.
- nucleic acid molecules which encode immunogenic portions of disease-associated antigens.
- antigens are deemed to be "disease-associated” if they are either associated with rendering a cell (or organism) diseased, or are associated with the disease-state in general but are not required or essential for rendering the cell diseased.
- antigens are considered to be “immunogenic” if they are capable, under appropriate conditions, of causing an immune response (either cell-mediated or humoral). Immunogenic "portions" may be of variable size, but are preferably at least 9 amino acids long, and may include the entire antigen.
- disease-associated antigens are contemplated within the scope of the present invention, including for example immunogenic, non- tumorigenic forms of altered cellular components which are normally associated with tumor cells.
- altered cellular components which are normally associated with tumor cells include ras (wherein is understood to refer to antigens which have been altered to be non-tumorigenic) and p53 .
- Other disease- associated antigens which may be encoded by the nucleic acid molecules provided herein include all or portions of various eukaryotic (including for example, parasites), prokaryotic (e.g., bacterial) or viral pathogens.
- viral pathogens include the Hepatitis B Virus ("HBV”) and Hepatitis C Virus (“HCV;” see U.S. Application No. 08/102/132), Human Papilloma Virus (“HPV;” see WO 92/05248; WO 90/10459; EPO 133,123), Epstein-Barr Virus (“EBV;” see EPO 173.254; JP 1,128,788; and U.S. Patent Nos. 4,939,088 and 5,173,414), Feline Leukemia Virus ("FeLV;” see U.S. Application No.
- Nucleic acid molecules of the present invention also include "Immunomodulatory cofactors", or, “immune accessory molecules” Briefly, these terms refers to molecules which can either increase or decrease the recognition, presentation or activation of an immune response (either cell-mediated or humoral).
- immune accessory molecules include IL-1, IL-2, IL-3, IL-4, IL-5, IL-6.
- IL-7 U.S. Patent No. 4,965,195
- IL-8, IL-9, IL-10, IL-11, IL-12 Wangf et al., J. Immun.
- heterologous gene encodes gamma-interferon.
- Nucleic acid molecules of the present invention also include those sequence which encode proteins that are required to "replace” a normal gene function.
- replacement genes refers to a nucleic acid molecule which encodes a therapeutic protein that is capable of preventing, inhibiting, stabilizing or reversing an inherited or noninherited genetic defect. Representative examples of such genetic defects include disorders in metabolism, immune regulation, hormonal regulation, and enzymatic or membrane associated structural function.
- Cystic Fibrosis due to a defect in the Cystic Fibrosis Transmembrane Conductance Regulator ("CFTCR"), see Dorin et al., Nature 326:6X4, Parkinson's Disease, Adenosine Deaminase deficiency ("ADA;” Hahma et al., J. Bad.
- CFTCR Cystic Fibrosis Transmembrane Conductance Regulator
- Nucleic acid molecules which encode the above-described sequences may be readily obtained from a variety of sources.
- plasmids which contain sequences that encode immune accessory molecules may be obtained from a depository such as the American Type Culture Collection (ATCC, Manassas, Virginia), or from commercial sources such as British Bio-Technology Limited (Cowley, Oxford England).
- Representative sources sequences which encode the above-noted immune accessory molecules include BBG 12 (containing the GM-CSF gene coding for the mature protein of 127 amino acids), BBG 6 (which contains sequences encoding gamma interferon), ATCC No. 39656 (which contains sequences encoding TNF), ATCC No.
- known cDNA sequences which encode a desired molecule may be obtained from cells which express or contain such sequences. Briefly, within one embodiment mRNA from a cell which expresses the gene of interest is reverse transcribed with reverse transcriptase using oligo dT or random primers. The single stranded cDNA may then be amplified by PCR (see U.S. Patent Nos. 4,683,202, 4,683,195 and 4,800,159. See also PCR Technology: Principles and Applications for DNA Amplification, Erlich (ed.), Stockton Press, 1989 all of which are incorporated by reference herein in their entirety) utilizing oligonucleotide primers complementary to sequences on either side of desired sequences.
- a double stranded DNA is denatured by heating in the presence of heat stable Taq polymerase, sequence specific DNA primers. ATP, CTP, GTP and TTP. Double-stranded DNA is produced when synthesis is complete. This cycle may be repeated many times, resulting in a factorial amplification of the desired DNA.
- Sequences which encode the above-described genes may also be synthesized, for example, on an Applied Biosystems Inc. DNA synthesizer (e g. , ABI DNA synthesizer model 392 (Foster City, California)).
- the nucleic acid molecules are provided in a composition in the form of a vector which directs the expression of a nucleic acid molecule of interest.
- the vector may direct the expression of more than one nucleic acid molecules.
- Such multiple sequences may be controlled either by a single promoter, or preferably, by additional secondary promoters (e.g., Internal Ribosome Binding Sites or "IRBS").
- IRBS Internal Ribosome Binding Sites
- a gene delivery vehicle directs the expression of heterologous sequences which act synergistically.
- vectors which direct the expression of a molecule such as IL-12, IL-2, gamma interferon, or other molecule which acts to increase cell-mediated presentation in the T H 1 pathway, along with an immunogenic portion of a disease- associated antigen.
- a molecule such as IL-12, IL-2, gamma interferon, or other molecule which acts to increase cell-mediated presentation in the T H 1 pathway, along with an immunogenic portion of a disease- associated antigen.
- immune presentation and processing of the disease-associated antigen will be increased due to the presence of the immune accessory molecule.
- compositions of the present invention are described that provide a greatly increased level of expression when the composition is administered in vivo.
- compositions which comprise a recombinant or synthetic nucleic acid molecule in an aqueous solution comprising 0.5% to 5% (w/v) polyalkylene glycol, or in a lyophilized composition comprising polyalkylene glycol.
- the polyalkylene glycol may be limited to a selected molecular weight (e.g., an average molecular weight of between about 1.000 and about 3,000, 4,000, 5,000, 6,000, 7,000, or, 8,000 daltons), or to a particular species or combination of species of polyalkylene glycols (e.g.. polypropylene glycol, polyethylene glycol, or, methoxy-polyethylene glycol).
- the composition comprises a recombinant or synthetic nucleic acid molecule in an aqueous solution comprising 0.5% to 5% (w/v) neutral or negatively charged polysaccharide, or in a lyophilized composition comprising a neutral or negatively charged polysaccharide.
- the neutral or negatively charged polysaccharide may be limited to a selected molecular weight (e.g., an average molecular weight of between about 1,000 and about 3,000, 4,000, 5,000, 6,000, 7,000, or, 8,000 daltons), or to a particular species or combination of species of polysaccharides (e.g., mannan, dextran (such as maltodextin), hyaluronic acid, or a glycosaminoglycan such as heparan sulfate, chondroitin sulfate or dermatan).
- a selected molecular weight e.g., an average molecular weight of between about 1,000 and about 3,000, 4,000, 5,000, 6,000, 7,000, or, 8,000 daltons
- mannan such as maltodextin
- hyaluronic acid such as maltodextin
- a glycosaminoglycan such as heparan sulfate, chondroitin sulf
- compositions of the present invention can optionally include other or additional pharmaceutically acceptable excipients.
- excipients can be used as fillers, processing aids, other delivery enhancers and modifiers, and the like.
- Suitable excipients include, for example, calcium phosphate, magnesium stearate, talc, monosaccharides, dissaccharides, polysaccharides, dextrose, low melting waxes, ion exchange resins, and the like, as well as combinations of any two or more thereof.
- a thorough discussion of pharmaceutically acceptable excipients is available in "Remingto 's Pharmaceutical Sciences” (Mack Pub. Co., NJ 1991).
- compositions can also be included in the compositions, such as, for example, marker agents, nutrients, and the like.
- agents that promote endocytosis of the desired nucleic acids or aid in binding of the nucleic acids to the cell surface, or both, can be incorporated into compositions of the present invention.
- Liquid compositions of the present invention can be in the form of a solution, suspension, or emulsion with a liquid carrier.
- suitable liquid carriers include, for example, water, saline, pharmaceutically acceptable organic solvent(s), pharmaceutically acceptable oils or fats, mixtures thereof, and the like.
- the liquid carrier may contain other suitable pharmaceutically acceptable additives, such as solubilizers, emulsifiers, nutrients, buffers, preservatives, suspending agents, stabilizers, and the like.
- Suitable organic solvents include, for example, monohydric alcohols, such as ethanol, and polyhydric alcohols, such as glycols.
- Suitable oils include, for example, soybean oil, coconut oil, olive oil, safflower oil, cottonseed oil, and the like.
- the carrier can also be an oily ester such as ethyl oleate, isopropyl myristate, and the like.
- the present invention provides methods for delivering nucleic acid molecules to an animal, comprising the general step of administering to an animal one of the nucleic acid molecule containing compositions described herein.
- Direct administration to an animal or group of cells within an animal can typically be accomplished by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly or delivered to an organ or tissue or to the interstitial space of an organ or tissue.
- the compositions can also be administered into a tumor or lesion.
- Other modes of administration include oral and pulmonary administration, rectal or vaginal suppositories or creams, and transdermal applications, needles, and gene guns or hyposprays, including nasal, oral or other mucosal sprays. Administration can also be by inplants, pumps, catheters or patches.
- Modes of administration include, for example, ex vivo administration to samples derived from an animal and in vitro administration to the sample. The sample can then be readministered to the animal after the cells have contacted and taken up the nucleic acid.
- the term animal includes all living organisms including eukaryotes and prokaryotes, vertebrates and invertebrates, reptiles, birds, and mammals, and within the category of mammals, includes rodents and humans among other subcategories.
- the compositions of the invention can be administered to any animal or to a group of cells or to a tissue from an animal or in an animal.
- tissues to which the compositions of the present invention may be delivered include tissue from mammals, such as, muscle, skin, brain, lung, liver, spleen, blood, bone marrow, thymus, heart, lymph, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, connective, and the like.
- the target tissue for the administration will be selected based on the nucleic acid to be taken up by the cells of the tissue and the goals of the therapy or administration.
- Cells suitable for use in the practice of the present invention include any cell inside an organism or animal, including mammals, particularly humans.
- Cell lines can also be used for the invention, for example, mammalian cell lines available from the American Type Culture Collection (ATCC), Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), human embryonic kidney cells, baby hamster kidney cells, mouse sertoli cells, canine kidney cells, buffalo rat liver cells, human lung cells, human liver cells, mouse mammary tumor cells, and other mammalian (including human) cells (e.g., stem cells, particularly hemopoietic cells, lymphocytes, macrophages, dendritic cells, tumor cells and the like).
- ATCC American Type Culture Collection
- BHK baby hamster kidney
- COS monkey kidney cells
- human hepatocellular carcinoma cells e.g., Hep G2
- compositions of the present invention can be administered in order to treat a particular condition or disease.
- the composition is administered in a therapeutically effective amount.
- a "therapeutically effective amount” should be understood to refer to those amounts which result in a physiologically significant effect.
- An effective dosage of nucleic acid administered directly to a subject will, of course, vary depending upon known factors such as the pharmacodynamic characteristics of the particular nucleic acid and encoded polypeptide, the mode and route of administration; the age, health, and weight of the recipient; nature and extent of symptoms; kind(s) of concurrent treatment, frequency of administration, the effect desired, and the like.
- compositions of excipients and nucleic acids can be administered as a single dose or in multiple doses. Multiple doses can be administered either continuously, in intervals, or a combination of both.
- an effective in vivo amount of nucleic acid will be from about 0.01 mg/kg to about 50 mg/kg or about 0.05 mg/kg to about 10 mg/kg of nucleic acid.
- Enhanced nucleic acid uptake by cells can be detected by using protein expression assays (where the nucleic acid encodes a polypeptide) or polynucleotide hybridization techniques (for either coding or noncoding nucleic acids).
- Compositions can be screened and optimized with respect to transfection efficiency by incorporating a reporter gene into the DNA and assaying for the reporter gene product using standard immunoassay methods or biological or enzymatic activity assays (such as, for example, a luciferase assay).
- the concentration of nucleic acid in stable preparations of excipient and nucleic acid is greater than the nucleic acid concentration of the dilute nucleic acid solution, and is typically at least about 150 ⁇ g (polynucleotide)/ml (preparation). More typically, the concentration of polynucleotide in stable preparation is at least about 250 ⁇ g/ml, more typically at least about 500 ⁇ g/ml, and even more typically at least about 1 mg/ml, and even more typically at least about 2 mg/ml.
- DNA plasmids containing a cytomegalovirus (CMV) promoter and chloramphenicol acetyltransferase (CMV-km-CAT), luciferase (CMV-km-Luc) or, ⁇ -galactosidase (CMV-km- or ⁇ -gal) reporter genes were prepared and purified.
- CMV cytomegalovirus
- CMV-km-CAT chloramphenicol acetyltransferase
- luciferase CMV-km-Luc
- ⁇ -galactosidase CMV-km- or ⁇ -gal reporter genes
- mice Five to six week old female Balb-c mice (20-25 grams, from Charles River) were used in all the animal studies. The mice were anesthetized by intraperitoneal administration of Ketamine/Xylazine cocktail. Plasmid DNA (25 or 50 ug) in 50 ul of the formulation was injected into the tibialis muscle of both legs after shaving the hair around the muscle. Injection depth was limited to 2 mm by inserting a plastic collar to the needle. Naked DNA injections in saline (plasmid DNA + saline) served as controls. Group sizes ranged from 3 to 10. In cases were large animal numbers were used to assess the variations in the data, pooled information from different experiments was used.
- luciferase activity was assayed using an automated Dynatech model ML2250 microplate luminometer. The amount of CAT protein was assayed by CAT ELISA follov ing manufacturer's instructions.
- Results are shown in Figures 1, 2 and 3. Briefly, enhanced luciferase gene expression was observed in initial in vivo experiments (Figure 1) using plasmid DNA encoding luciferase gene injected into mouse TA muscles with 2% PEG 2000, 2% maltodextrin or 2% hyaluronic acid. Results shown in Figure 1 are pooled information obtained from DNA injections in 13 muscles. Direct injection of a plasmid DNA (50 ug in saline) into mouse TA muscle produced an average of 4.9 ng of luciferase per muscle at 8 days post injection.
- Formulations were prepared fresh on the day of injection. Formulations were made by aliquoting appropriate volumes of stock solutions in the following order: water, 5M NaCl, excipient, and pDNA to obtain a final pDNA concentration of 1.5 ⁇ g/ ⁇ l or 0.75 ⁇ g/ ⁇ l in 150 mM saline with 2% Maltodextrin.
- C57/BL6 mice were anesthetized by intraperitoneal administration of Ketamine/Xylazine cocktail. Baseline hematocrit were measured before injection of DNA formulation. Plasmid DNA (see U.S. Application No. 08/910,647, filed August 13, 1997) in 50 ⁇ l of the formulation was injected into the tibialis muscle of both legs. Injection depth was limited to 2 mm by inserting a plastic collar to the needle.
- serum was recovered from the capillary tubes and stored at -20C for the measurement of EPO concentration.
- Serum concentrations of monkey EPO were determined by ELISA system (Quantikine IVD, R&D systems, Minneapolis, MN) using a mouse monoclonal antibody according to the manufacturer's protocol. D. Results
- Results are shown in Figure 4. Briefly, while DNA in saline (at concentrations of 75 or 150ug) do show expression after 1 or 2 weeks, administration of DNA in maltodextin shows an increase of at least 2 to 4-fold in the quantity of EPO expressed.
- DNA encoding EPO in a 2% (w/v) maltodextrin formulation elevated the hematocrit to at least 10% higher level compared to DNA administration in saline.
- a dose response study showed that mice injected with 50 ⁇ g DNA in maltodextrin formulation can reach the same hematocrit levels as mice injected with 100 ⁇ g DNA in saline formulation.
- DNA plasmids containing a CMV promoter and EPO gene were prepared as described above.
- Maltodextrin (dextrose equivalent 13.0-17.0) was obtained from Aldrich (Milwaukee, WI).
- EPO Elisa kit was purchased from R&D systems (Minneapolis, MN).
- Formulations were made by aliquoting appropriate volumes of stock solutions in the following order: water, 5M NaCl, 10% maltodextrin, and pDNA to obtain a final pDNA concentration of 0.2 ⁇ g/ ⁇ l ⁇ 2 ⁇ g/ ⁇ l in 150 mM saline with or without 2% (w/v) maltodextrin.
- Example Preparation of 350 ⁇ l of DNA formulation in 2% (w/v) maltodextrin at 1 mg/ml concentration.
- mice Balb/c, C57/BL6 or SCID Beige mice were used in the experiments.
- the mice were anesthetized by intraperitoneal administration of Ketamine/Xylazine cocktail.
- Plasmid DNA (10- 100 ⁇ g) in 50 ⁇ l of the formulation was injected into the tibialis muscle of both legs. Injection depth was limited to 2 mm by inserting a plastic collar to the needle. Hematocrits were determined periodically by centrifugation of blood in a micro-capillary tube. Serum EPO levels were assayed by ELISA using the human EPO Quantikine IVD kit from R & D systems according to manufacturer's recommendations.
- Biological activity of the recombinant EPO is shown by the elevation of hematocrit in the experimental animals.
- C57/BL6 mice were injected intramuscular with 25, 50 or 100 ⁇ g of EPO plasmid in formulations containing either 2% maltodextrin or saline. Maltodextrin in saline served as buffer control. Hematocrits were monitored periodically during the 60 days after injection. Preinjection values of hematocrit were around 48%. Injections of control buffer alone did not result in any elevation of the hematocrit levels. Injections of low dose formulations (25 ⁇ g) elevated hematocrit levels very minimally.
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AU39924/99A AU3992499A (en) | 1998-05-15 | 1999-05-14 | Compositions and methods for the delivery of nucleic acid molecules |
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US8558798P | 1998-05-15 | 1998-05-15 | |
US60/085,587 | 1998-05-15 | ||
US31160299A | 1999-05-13 | 1999-05-13 | |
US09/311,602 | 1999-05-13 |
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WO1999059638A2 true WO1999059638A2 (en) | 1999-11-25 |
WO1999059638A3 WO1999059638A3 (en) | 2000-02-24 |
WO1999059638A8 WO1999059638A8 (en) | 2000-08-03 |
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US8241655B2 (en) * | 2004-05-12 | 2012-08-14 | Surmodics, Inc. | Coatings for medical articles including natural biodegradable polysaccharides |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5410016A (en) * | 1990-10-15 | 1995-04-25 | Board Of Regents, The University Of Texas System | Photopolymerizable biodegradable hydrogels as tissue contacting materials and controlled-release carriers |
WO1996021470A2 (en) * | 1995-01-13 | 1996-07-18 | Genemedicine, Inc. | Compositions of nucleic acid and viscosity-increasing polymers for use in gene therapy |
WO1997005185A2 (en) * | 1995-07-28 | 1997-02-13 | Focal, Inc. | Multiblock biodegradable hydrogels for use as controlled release agents for drugs delivery and tissue treatment agents |
WO1998000112A1 (en) * | 1996-07-02 | 1998-01-08 | University College Dublin | Organised assemblies containing entrapped negatively charged polyelectrolytes |
WO1999006026A1 (en) * | 1997-07-30 | 1999-02-11 | Biovector Therapeutics (S.A.) | Stable particulate complexes with neutral or negative global charge of lamellar structure |
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1999
- 1999-05-14 WO PCT/US1999/010697 patent/WO1999059638A2/en active Application Filing
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5410016A (en) * | 1990-10-15 | 1995-04-25 | Board Of Regents, The University Of Texas System | Photopolymerizable biodegradable hydrogels as tissue contacting materials and controlled-release carriers |
WO1996021470A2 (en) * | 1995-01-13 | 1996-07-18 | Genemedicine, Inc. | Compositions of nucleic acid and viscosity-increasing polymers for use in gene therapy |
WO1997005185A2 (en) * | 1995-07-28 | 1997-02-13 | Focal, Inc. | Multiblock biodegradable hydrogels for use as controlled release agents for drugs delivery and tissue treatment agents |
WO1998000112A1 (en) * | 1996-07-02 | 1998-01-08 | University College Dublin | Organised assemblies containing entrapped negatively charged polyelectrolytes |
WO1999006026A1 (en) * | 1997-07-30 | 1999-02-11 | Biovector Therapeutics (S.A.) | Stable particulate complexes with neutral or negative global charge of lamellar structure |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8241655B2 (en) * | 2004-05-12 | 2012-08-14 | Surmodics, Inc. | Coatings for medical articles including natural biodegradable polysaccharides |
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WO1999059638A3 (en) | 2000-02-24 |
WO1999059638A8 (en) | 2000-08-03 |
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