WO1997045069A1 - Cationic polymer/lipid nucleic acid delivery vehicles - Google Patents
Cationic polymer/lipid nucleic acid delivery vehicles Download PDFInfo
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- WO1997045069A1 WO1997045069A1 PCT/US1997/009275 US9709275W WO9745069A1 WO 1997045069 A1 WO1997045069 A1 WO 1997045069A1 US 9709275 W US9709275 W US 9709275W WO 9745069 A1 WO9745069 A1 WO 9745069A1
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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
- A61K9/10—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
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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
- A61K47/50—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
- A61K47/56—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
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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
- A61K47/50—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
- A61K47/56—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/595—Polyamides, e.g. nylon
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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
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
- C08G73/02—Polyamines
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
- C08G73/02—Polyamines
- C08G73/0206—Polyalkylene(poly)amines
- C08G73/0213—Preparatory process
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- 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
Definitions
- the present invention is in the field of biochemistry.
- novel compositions and methods are reported which efficiently deliver polynucleotides or other bioactive materials to cells.
- the present invention relates to novel cationic polymers and cationic lipids that are useful for the delivery of polynucleotides to cells.
- Polynucleotides are typically engineered to perform a specific function within the cell.
- polynucleotide polymers are highly charged molecules (due to the phosphate backbone) and do not readily permeate the cell membrane.
- liposomes The phospholipid bilayer of the liposome is typically made of materials similar to the components of the cell membrane.
- polynucleotides associated with liposomes may be delivered to the cell when the liposomal envelope fuses with the cell membrane. More typically, the liposome will be endocytosed into the cell.
- the internal pH of the endocytic vesicle may drop substantially, and/or the vesicle may fuse with other intracellular vesicles, including lysosomes.
- the internal contents of the endosome may be released into the cell.
- Liposomes are limited as polynucleotide delivery vehicles by the relatively small internal volume of the liposome. Thus, it is difficult to effectively entrap a large concentration of polynucleotide within a liposomal formulation.
- Patent No. 5,208,036 to Epstein et al . ) Patent No. 5,208,036 to Epstein et al . ) ; TRANSFECTAM TM (DOGS) a synthetic cationic lipid with lipospermine head groups (Promega, Madison, Wisconsin) ; DMRIE and DMRIE ⁇ HP (Vical, La Jolla, CA) ,- DOTAP TM (Boehringer Mannheim (Indianapolis, Indiana) , and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Maryland) .
- TRANSFECTAM TM DOGS
- DMRIE and DMRIE ⁇ HP Vical, La Jolla, CA
- DOTAP TM Boehringer Mannheim (Indianapolis, Indiana)
- DOSPA Lipofectamine
- the above compounds enhance the permeability of nucleic acids to cells cultured in vi tro , and the process of lipofection has become an important tool of cellular biology.
- formulations comprising the cationic _lipids are intermixed with the polynucleotide to be delivered and then applied to the target cells.
- the cationic lipid-polynucleotide complex must generally be used relatively soon after mixing because lipofection efficiency rapidly decreases to undectable levels 24 hours after formulation. From this observation, one may surmise that, at least with respect to lipofection efficiency, the cationic lipid-polynucleotide complex is rather unstable, or has an extremely limited shelf life.
- Another draw-back of the presently available synthetic cationic lipids is that the respective lipid and cationic components are not joined by a biodegradable chemical linkage which presumably contributes to the inherent toxicity of the synthetic cationic lipids.
- a given level of cellular toxicity may be detrimental but acceptable where in vi tro, or research, use of cationic lipids to deliver polynucleotides is contemplated; however, such toxicity is generally unacceptable where in vivo use of cationic lipids is contemplated.
- synthetic cationic lipids which comprise biocompatible, biodegradable, or metabolizable components would be preferred for the preparation of cationic lipid-polynucleotide delivery vehicles for use in vivo .
- lipid groups may be joined to suitable _cationic components in an attempt produce cationic lipids with reduced toxicity.
- the toxicity of the synthetic cationic lipids may be reduced by assembling the cationic lipids into suitably constructed polynucleotide delivery vehicles.
- the present invention relates to a novel class of synthetic biocompatible cationic lipids and cationic polymers that are useful for polynucleotide packaging and delivery.
- the present invention describes the use of a combination of primary and secondary amines separated by, for example, ethylene hydrocarobons (i.e., multi-valent cationic groups, such as pentaethylenehexamine (PEHA) ) , to derivatize suitable lipid groups, e.g., phospholipid, cholesterol, etc..
- PEHA pentaethylenehexamine
- an embodiment of the present invention is the novel compound triaminocholesterol (TAC) which comprises a PEHA derivative (diethylene triamine) covalently linked to cholesterol .
- TAC triaminocholesterol
- the multivalent cationic groups may be assembled into cationic polymers.
- the cationic polymers of the present invention are comprised of substantially biocompatible cationic monomers that are interconnected by a biocompatible or substantially biocompatible linking groups.
- the chemical linkages used to construct the cationic polymers of the present invention are hydrolyzable under physiological conditions or, more preferably, are biodegradable.
- the cationic moieties are linked by biocompatible covalent bonds such as a disulfide bonds, hydrolyzable bonds, pH sensitive bonds, or any combination thereof .
- inventions include stable polynucleotide delivery vehicles comprising the novel cationic lipids and/or cationic polymers, and methods for producing and using the same.
- R 1 and R 2 are drawn from the group consisting of H, C 1 -C 6 alkyls, alkenyls, or alkynyls, or mono or multivalent cationic amine groups (e.g., spermine, spermidine, pentaethylenehexamine (PEHA) , diethylene triamine, pentamethylenehexamine, pentapropylenehexamine, etc.)
- Y is a cholesterol or cholesterol derivative.
- R is a cationic group capable of binding nucleic acid
- X is a biocompatible, biodegradable or otherwise labile covalent cross-linker molecule
- n is number between about ten and up to about ten thousand.
- a preferred average molecular weight for polymer preparations will typically be between about 40,000 daltons and about 1,000,000, more typically between about 60,000 daltons and about 250,000 daltons, preferably between about 80,000 daltons and about 150,000 daltons, and more preferably between about 90,000 daltons and about 110,000 daltons.
- R may comprise any one of a group of cations that are used to make a heteropolymer .
- heteropolymeric cations are contemplated which have the general structure: ( ... (X-R x -X-R y -X) ... ) wherein R x is a given cation that is capable of interacting with nucleic acid; R y is any of a number of cations other then R x that is also capable of interacting with nucleic acid; and X is a biocompatible cross-linker molecule.
- Another embodiment of the present invention includes a novel process for making polynucleotide delivery vehicles comprising the steps of complexing the polynucleotide and cationic polymer and/or cationic lipid in buffer that maintains DNA as a B-form helix (e.g., an aqueous alcohol solution) , and removing the buffer by evaporation. After reconstitution of the dried polynucleotide-cationic lipid/cationic polymer complex with aqueous solution, stable polynucleotide delivery vehicles are produced.
- buffer e.g., an aqueous alcohol solution
- Another embodiment of the present invention contemplates the use of polynucleotide delivery vehicles comprising cationic lipids and/or polymers to deliver a polynucleotide, or polynucleotides, of interest to a cell. Accordingly, the described cationic polymers may be used to provide a therapeutic benefit to the individual .
- FIGURES show how and where representative cationic groups may be polymerized using an appropriate dicarboxylic acid linker molecule.
- Figure 2 provides additional examples of several alternative cationic groups and linking agents that may be used to produce cationic polymers .
- Figure 3 shows a schematic synthesis scheme for the production of a novel cationic phospholipid.
- Figure 4 shows a schematic synthesis scheme for triaminocholesterol .
- Figure 5 shows in vivo expression data obtained using the PDVs produced by the ethanol evaporation method.
- biocompatible cationic polymers or cationic lipids of the present invention may be contacted (ion paired) with a polynucleotide, or polynucleotides, of interest such that the positive charge of the cationic groups electrostatically interacts with the negatively charged polynucleotide.
- the electrostatic interaction between the cationic moiety and the polynucleotide presumably reduces charge repulsion in the polynucleotide and allows the polynucleotide to be condensed into a more compact configuration (as seen by gel-shift assays, etc. ) .
- the cationic component used in the presently described cationic lipids and polymers may be monovalent, divalent, multivalent, or preferably polyvalent (i.e., polycationic) .
- monovalent cations capable of associating with DNA include primary amines, including, but not limited to methylamine, ethylamine, etc.) , and multivalent amines such as, but not limited to, spermine, spermidine, pentaethylenehexamine, diethylene triamine, pentamethylenehexamine, pentapropylenehexamine.
- the cationic component is preferably biocompatible or biotolerable.
- the cationic component may comprise any of a variety of chemical groups that retain a positive charge between pH 5 through pH 8 including, but not limited to, amino groups (or oligo or poly amines) , e.g., spermine, spermidine, pentaethylenehexamine (PEHA) , diethylene triamine, pentamethylenehexamine, pentapropylenehexamine, etc.) , amide groups, amidine groups, positively charged amino acids (e.g. , lysine, arginine, and histidine) , imidazole groups, guanidinium groups, or mixtures and derivatives thereof.
- amino groups or oligo or poly amines
- the cationic component will generally be combined with the polynucleotide at a cation/phosphate ratio that has been optimized for a given application.
- the cation/phosphate ratio will be between about 0.1 and about 20, often between about 5 and about 17, and preferably between about 6 and about 15.
- the charge ratio will vary accordingly depending on the number of positively charged groups co . ntained on the cation, and the size of the polynucleotide.
- DOSPA/DNA nucleotide ratio of about 0.6 is suitable. Because of the inherent toxicity of presently available cationic lipids, i.e., DOSPA, such lipids are generally not preferred for in vivo gene delivery.
- cationic lipids having reduced toxicity are preferred facilitators of nonviral delivery of polynucleotides in vivo .
- additional embodiments of the present invention are novel cationic lipids produced by reacting multivalent cationic (amino) groups with, for example, cholesterol or DOPE.
- a preferred embodiment of the present invention is a compound having the formula:
- n is about 1 to about 6, and R is drawn from the group consisting of:
- R x and R 2 are drawn from the group consisting of H, methyl, ethyl, —C j -C 4 alkyls, alkenyls, or alkynyls, -
- a particular example of one such cationic lipid includes the cationic phospholipid produced essentially as shown in
- R is drawn from the group consisting of:
- R x and R 2 are drawn from the group consisting of H, methyl, ethyl, —C 1 -C 4 alkyl, alkenyl, or alkynyls, -(CH 2 ) n NH 2 , -(CH 2 ) n NH(CH 2 )
- a particular example of one such cationic lipid includes the molecule triaminocholesterol (TAC) .
- TAC was constructed by reacting a diethylene triamine derivative of PEHA to a suitably treated cholesterol derivative essentially as shown in Figure 4.
- cross-linking agents used to prepare the presently described polymers are preferably biocompatible or biotolerable, and will generally comprise at least two chemical groups (i.e. , the cross- linkers are bifunctional) that are each capable of forming a bond with a suitable chemical group on the cation.
- the linker groups may be homobifunctional (same chemical groups) or heterobifunctional (different chemical groups) .
- the chemical linkage formed between the linking group and the cationic moiety will be hydrolyzable under physiological conditions (i.e., pH labile, or otherwise subject to breakage in the target cell) .
- the cross-linking agent may comprise a bond that is hydrolyzable under physiological conditions in between the linking groups.
- the cross-linking agent may be combined with an additionally cross-linking agent that a allows for the formation of branched polymers.
- the cationic and linker components of the claimed cationic polymers are described in, or may be obtained from any of a variety of sources including, but not limited to, the 1995 edition of the Merck Index, Budavari, et al . , eds. , Merck and Company, Inc, Rahway, N.J., the 1995 SIGMA chemical company catalogue, St. Louis, MO., the 1995 Aldrich Biochemicals Catalogue, or the 1995 Ofatlz and Bauer catalogue.
- the cationic group may preferably be attached to the cross-linker by an amide, ester, or phosphodiester linkage which renders the linker separable from the cationic group under physiological conditions or by the action of natural enzymes such as glycosylases, proteases, lipases or phospholipases, and the like.
- Such a linkage represents an improvement over the currently available synthetic cationic lipids which are inherently toxic.
- An additional feature of the presently described polymerization reaction is that, preferably, practically useful cationic polymers may be formed without strictly requiring the employment of protecting groups, or elaborate deprotecting schemes.
- One embodiment of the present invention is the use of linker molecules that are at least multicarboxylic acid derivatives of carbohydrates to form cationic polymers.
- the molecules will be at least dicarboxylic derivatives of carbohydrates (i.e., mono, di, or polysaccharide molecules) , and will cross-link the cationic moieties by amide linkages.
- polymeric carbohydrates i.e., similar to murein
- the cationic group is a polyamine
- any compound comprising dicarboxylic acid groups may act a_s a suitable linker molecule.
- the linkers will be soluble under aqueous conditions, and the carboxylic acid groups will generally have a least one to three carbon atoms interspersed between the groups .
- dicarboxylic acids that incorporate additional groups that increase hydrophilicity, while not substantially interfering with the polymerization reaction (i .e. ,hydroxyl groups or poly ethers) .
- Additional linker molecules include the general type, or molecules employing a similar chemical strategy, as those described in U.S. Patent No. 4,833,230 are herein incorporated by reference.
- linkers that form acid labile bonds upon reaction with amino groups.
- pH labile bonds comprise working exemplifications of the claimed pH sensitive/labile covalent linker moieties (which may also include ester linkages) .
- biodegradable cationic polymer shall refer to the fact that upon entering into the cell the cationic polymer is converted to components (and metabolizable byproducts thereof) that are generally capable of participating in the catabolic or metabolic processes of the cell, or are excreted by the cell and voided.
- biocompatible shall mean that the compound does not display significant toxicity or adverse immunological effects at the contemplated dosages.
- biotolerable shall mean that an item or compound may be used to treat animals or animal cells with manageable side- effects or toxicity effects.
- pH sensitive shall mean that at least one covalent bond in the molecule may be broken by a change in pH that generally approximates that which occurs after endosomal fusion.
- substantially toxic shall mean that, at therapeutic dosages, a given agent produces harmful consequences which, on balance, clearly outweigh the contemplated therapeutic benefits of the agent.
- Another method of polymerizing spermine, PEHA, or other cations, jusing a biodegradable linkage involves using dipeptide linkers which are susceptible to proteolytic cleavage by lysosomal proteases, including, but not limited to, thioproteases or cathepsins.
- Additional embodiments of the present invention are novel methods of using the above-described cationic polymers and cationic lipids to deliver polynucleotides to cells in vi tro or in vivo.
- the cationic polymers and cationic lipids may be used in conjunction with conventional lipids, or currently available cationic lipid conjugates (e.g., Lipofectin, Lipofectamine, and the like) .
- the gene delivery is conducted using a method that is substantially nontoxic to the cells or patient.
- the presently described cationic polymers will generally form structures in aqueous solution that are characteristic of a given polymer.
- the polymers form a relatively compact structure in water, swell in the presence of added salt, and form an intermediate sized structure when polynucleotide is added.
- the changes in the physical size and density of the molecule before and after polynucleotide association allow one to follow the progress of polynucleotide association, and facilitate the isolation of the desired product.
- Polynucleotide delivery vehicles comprising the disclosed cationic lipids or cationic polymers, or a mixture thereof, generally incorporate the polynucleotide to be delivered as a structural component of the PDV.
- the structure of the polynucleotide contributes to the structural characteristics of the PDV.
- the DNA will generally comprise either super-coiled or relaxed circles, or a mixture thereof.
- enzymes such as DNA gyrase, ligase, and topoisomerase may be used to alter the structure of the plasmid as deemed necessary.
- plasmids may be linearized, and optio_nally concatamerized, prior to complex formation.
- Single- and double-stranded polynucleotides might also be "prepackaged" prior to complex formation by the addition of suitable polynucleotide binding proteins such as viral proteins, single-stranded binding protein, histone proteins and the like.
- Polynucleotides of interest that may be delivered using the claimed polynucleotide delivery vehicles include, but are not limited to, DNA, RNA, polynucleotides associated with procaryotic and eucaryotic viral particles (e.g., retroviral core particles, bacteriophage particles, adenovirus particles, adenoassociated virus core particles, and the like) , protein/DNA complexes, i.e., proteins for integration, endosome disruption, to facilitate gene transfer and expression, etc.; RNA/DNA complexes, and any and all derivatives and variations of the above.
- a DNA molecule is to be delivered, it will typically comprise a gene of interest, or portion thereof, which is flanked by regulatory sequences which are spatially organized to optimize the expression of the DNA of interest.
- the polynucleotide to be delivered using the presently described PDVs will be substantially pure (i.e., substantially free of contaminating proteins, lipid, polysaccharide, lipopolysaccharide, and nucleic acid) .
- the preparations will generally be prepared by a process comprising phenol, or phenol :chloroform, extraction, and isopycnic centrifugation (using CsCl , and the like) , or functional equivalents thereof.
- the DNA preparations will also be treated with RNase, and subject to multiple rounds of extraction, and at least two rounds of ultracentrifugation (or any other means of producing DNA at least as pure) .
- a substantially pure preparation of nucleic acid is a preparation in which at least about eighty percent, generally at least about ninety percent, and preferably at least about ninety five percent of the total nucleic acid is comprised of the desired nucleic acid.
- Genes of interest are typically inserted into any of a wide range of expression vectors which are subsequently delivered using the presently disclosed methods and materials.
- Suitable vectors which may be delivered using the presently disclosed methods and compositions include, but are not limited to, herpes simplex virus vectors, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, pseudorabies virus, alpha-herpes virus vectors, and the like.
- compositions may be used to directly deliver vector nucleic acid, or, where applicable, viral or subviral particles encoding or containing the nucleic acid of interest.
- expression refers to the transcription of the DNA of interest, and the splicing, processing, stability, and, optionally, translation of the corresponding mRNA transcript . Depending on the structure of the DNA molecule delivered, expression may be transient or continuous.
- transcriptional promoters and enhancers may be 1 used in the DNA of interest, including, but not limited to, the herpes simplex thy ⁇ nidine kinase promoter, cytomegalovirus promoter/enhancer, SV40 promoters, and retroviral long terminal repeat (LTR) promoter/enhancers, and the like, as well as any permutations and variations thereof, which may be produced using well established molecular biology techniques (see generally, Sambrook et al . (1989) Molecular Cloning Vols . I-III, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, and Current Protocols in Molecular Biology (1989) John Wiley & Sons, all Vols.
- LTR long terminal repeat
- Promoter/enhancer regions may also be selected to provide tissue-specific expression.
- the genes of interest will also be engineered to comprise a suitable 3' polyadenylation sequence (if necessary) .
- DNAs of particular interest include, but are not limited to, sequences encoding a variety of proteins, cytokines and growth factors, (such as, G-CSF, GM-CSF, nerve growth factor (NGF) , ciliary neurotropic factor (CNTF) , brain-derived neurotropic factor (BDNF) , interleukins 1-2 and 4-14, tumor necrosis factor- ⁇ (TNF- ⁇ ) , or or ⁇ interferons, erythropoietin, and the like) , the cystic fibrosis transmembrane conductance regulator (CFTR) , tyrosine hydroxylase (TH) , D-amino acid decarboxylase, GTP cyclohydrolase, leptin, leptin receptor, factors VIII and IX, tissue plasminogen activator (tPA) .
- cytokines and growth factors such as, G-CSF, GM-CSF, nerve growth factor (NGF) , ciliary neurotropic factor (
- biologically active material includes, in particular, pharmaceutically active proteinaceous materials, and pharmaceutically active organic molecules.
- antisense, antigene, or aptameric oligonucleotides may be delivered using the presently described PDVs.
- PNAs polynucleotide peptide bonded oligos
- RNAs of interest include self-replicating RNAs, mRNA transcripts corresponding to any of the above genes which may be directly translated in the cytoplasm, or catalytic RNAs, e.g. "hammerheads" hairpins, hepatitis delta virus, group I introns which may specifically target and/or cleave specific RNA sequences in vivo .
- catalytic RNAs e.g. "hammerheads" hairpins, hepatitis delta virus, group I introns which may specifically target and/or cleave specific RNA sequences in vivo .
- RNA viruses as well as both cellular and viral transcripts.
- antisense forms of RNA, DNA, or a mixture of both may be delivered to cells to inhibit the expression of a particular gene of interest in the cell or to correct point, or other (nonsense or missense, etc.) mutations.
- An additional embodiment of the present invention contemplates the delivery of oligomeric nucleotides which have been incorporated into the PDVs in conjunction with larger polynucleotides.
- Such "carrier" polynucleotides may be single-stranded (linear or circular) , or substantially double-stranded, and may additionally comprise one or more regions which are substantially homologous or complementary to the oligomeric nucleotides to be delivered.
- the DNA of interest may further incorporate a suicide signal that allows for the controlled extermination of cells harboring and expressing the DNA of interest previously delivered by the delivery vehicle.
- the thymidine kinase (tk) gene may be incorporated into the delivered DNA which would allow the practitioner to subsequently kill cells expressing the tk gene by administering the correct amounts of acyclovir, gangcyclovir, or the conceptual or functional equivalents thereof.
- the presently described methods for producing polynucleotide delivery vehicles require that the polynucleotide (s) of interest be contacted with the amphipathic cationic lipid conjugates such that ion pairing between the cationic moiety and the polynucleotide allows for complex formation.
- the condensed cationic polymer/lipid- polynucleotide complex may subsequently serve as a scaffold, or nucleus, for the assembly of polynucleotide delivery vehicles (PDVs) .
- the cationic polymer/lipid- polynucleotide complex may be used directly.
- the pH during complex formation may be varied to optimize or stabilize the interaction of the specific components. For instance, where non-pH sensitive cationic polymers are used, a pH as low as about 4 may be preferred to complex a given polynucleotide (e.g., RNA) or other chemical agent which may be coincorporated with the polynucleotide. Additionally, where the polynucleotide (e.g., DNA) is not substantially sensitive to base hydrolysis, circumstances may dictate that a pH of up to about 10 be used during complex formation. Generally, a pH within the range of about 5 to about 9 will be maintained during complex formation and transfection.
- a pH as low as about 4 may be preferred to complex a given polynucleotide (e.g., RNA) or other chemical agent which may be coincorporated with the polynucleotide.
- the polynucleotide e.g., DNA
- a pH within the range of about 5 to about 9 will be maintained during complex formation and transfection.
- the concentration of salt e.g, NaCl, KC1, MgCl 2 , etc.
- concentration of salt may be varied to optimize complex formation, or to enhance the efficiency of gene delivery and expression.
- factors such as the temperature at which the cationic lipids or cationic polymers are complexed may be varied to optimize the structural and functional attributes of the resulting PDVs.
- the osmolarity of solution in which the complexes are formed may be altered by adjusting salt concentration.
- osmolarity may be adjusted by adding or substituting suitable excipients such as, but not limited to, glucose, sucrose, lactose, fructose, trehalose, maltose, mannose, and the like.
- suitable excipients such as, but not limited to, glucose, sucrose, lactose, fructose, trehalose, maltose, mannose, and the like.
- cationic condensing agents e.g., spermine, PEHA, or spermidine
- the carefully controlled addition of condensing cationic polymer to the polynucleotide allows for relatively high concentrations of polynucleotide (e.g., about 0.5 mg/ml) to be complexed with the condensing agent.
- carefully controlled addition of the polynucleotide to the cationic condensing agent allows for relatively high concentrations of polynucleotide to be complexed by the cationic condensing agent.
- the lipid is generally dissolved or solubilized in a suitable detergent in order to form lipid micelles.
- suitable detergents suitable for dissolving lipids include, but are not limited to cholate, deoxycholate, lauroyl sarcosine, octanoyl sucrose, CHAPS (3- [ (3-cholamidopropyl) -di-methylamine] -2-hydroxyl-l-propane) , novel- ⁇ -D-glucopyranoside, Lauryl dimethylamine oxide, octylglucoside, and the like.
- the detergent will be nonionic and possess a high critical micelle concentration (CMC) .
- CMC critical micelle concentration
- slow removal of the detergent i.e., by extensive dialysis
- slow dialysis remains the preferred method of detergent removal
- the polynucleotide and cationic polymer may be dissolved in a solution containing a suitable cation prior to the addition of lipid and detergent. After the detergent is added, it is removed by dialysis in the presence of cation, and subsequently the cation may removed by dialysis.
- suitable cations include any element carrying a positive charge.
- the cation may be monovalent, divalent, or multivalent .
- suitable elemental cations include, but are not limited to manganese, magnesium, sodium, calcium, rubidium, zinc, molybdenum, nickel, iron and the like.
- the elemental cation will be added in an amount sufficient to prevent aggregate formation during complexation of the lipid and the polynucleotide, and up to a concentration of about the maximum solubility of a given cationic compound.
- concentration of sodium, (e.g., sodium chloride) will be between about 0.1 molar and about 5 molar
- the concentration of magnesium (e.g., magnesium chloride) will be between about .05 molar and about 5 molar
- the concentration of manganese e.g., manganese chloride
- the type and concentration of cation may have to be adjusted depending on the characteristics of the cationic polymer used to assemble the PDVs.
- the cationic polymer or cationic lipid (molecular cations) , and/or detergent may be added prior to, concurrently with, or subsequent to, the addition of cation.
- the cationic polymer and cationic lipid will be added to the poly, or oligo, nucleotide at a net molecular cation-to- polynucleotide phosphate ratio of between about 0.1:1 and about 16:1, preferably between about 0.5:1 and about 7:1, more preferably between about 0.7:1 and about 2:1, and specifically about 1:1.
- the above ratios are provided for exemplification and not limitation, and may be modified depending on the characteristics of the molecular cations used to assemble the PDVs. Also, the optional ratio will be dependent upon the DNA concentration.
- the detergent will preferably be removed by dialysis in cation comprising buffer.
- the cation may subsequently be substantially removed by dialysis, or a functional equivalent.
- dialysis will generally be performed at a temperature of between about 4°C and about 30°C, and will result in a final cation concentration that is not detrimental to the intended use of the PDV.
- the elemental cation may be substantially removed by, for example, dialysis with a buffered solution that is suitable for parenteral administration.
- the resulting PDVs After the substantial removal of the cation, the resulting PDVs generally remain stable (i.e., retain transduction activity) for at least two weeks when stored at about 4°C, or may be lyophilized and stored indefinitely. Because the presently described cationic polymers are preferably biocompatible, PDVs comprising the cationic polymers will bear reduced toxicity. For the purposes of the present disclosure, reduced toxicity shall mean that PDVs comprising at least about 10 ⁇ g of DNA may be injected into an animal without the animal suffered grave toxicity effects.
- preparations of PDVs that have been formulated as described above and comprise a concentration of DNA (or other nucleotide) of generally between about .05 mg/ml and about 10 mg/ml, preferably between about .25 mg/ml and about 10 mg/ml, more preferably between about .5 mg/ml and about 1.5 mg/ml, and specifically between about .8 mg/ml and 1.2 mg/ml.
- a concentration of DNA or other nucleotide
- PDV compositions comprising high concentrations of nucleic acid (i.e. >.25 mg/ml nucleic acid) .
- Formulating PDVs using the detergent dialysis method described above typically generates particles that are greater than 200 nm in mean diameter. Where smaller particles may be preferred, an alternative method for formulating PDVs involves forming the cationic lipid- polynucleotide complex in a mixed aqueous solution that has been formulated to maintain the polynucleotide in a structural conformation that is conducive to binding by cationic lipids or cationic polymers. Examples of such solutions include mixed water/alcohol solutions (methanol, ethanol, isopropanol, butanol, and isomers and mixtures thereof) . Preferably, such complexation buffers also contain a concentration of dissolved sugar and/or salt in addition to a percentage of alcohol .
- the concentration of alcohol (e.g., ethanol) present during complex formation shall range from between about 10 . percent up to about 80 percent, typically between about 20 percent and about 50 percent, more typically between about 30 percent and about 45 percent, preferably between about 37 percent and about 43 percent, and more preferably about 40 percent.
- alcohol e.g., ethanol
- the amount of sugar (dextrose, sucrose, etc., see list provided above) that may be present during complex formation shall generally vary from between about 2 percent and about 15 percent, preferably between about 3 percent and about 8 percent, and more preferably about 5 percent.
- the osmolarity of the solution may also be adjusted by a mixture of salt and sugar.
- concentration of salt and sugar Typical concentrations of salt and sugar that may serve as a starting point for further optimization are 250 mM (glucose) and 25 mM salt (NaCl) .
- An additional feature of complex formation is temperature regulation.
- cationic lipids or polymers are complexed with polynucleotide at a temperature between about 4° C and about 65° C, more typically between about 10° C and about 42° C, preferably between about 15° C and about 37° C, and more preferably at about room temperature.
- the dry complex After the solution is removed from the complex by, for example, evaporation, the dry complex remains stable and may be stored indefinitely. After reconstitution, the size of the complex may be further adjusted by established means such as extrusion, homogenization, sonication, and the like.
- targeting agents may be stably incorporated into the vehicles to direct the vehicles to specific cells and/or tissues. Accordingly, any of a variety of targeting agents may be also be incorporated into the delivery vehicles .
- targeting agent shall refer to any and all ligands or ligand receptors which may be incorporated into the delivery vehicles.
- Such ligands may include, but are not limited to, antibodies such as IgM, IgG, IgA, IgD, and the like, or any portions or subsets thereof, cell factors, cell surface receptors, MHC or HLA markers, viral envelope proteins, peptides or small organic ligands, derivatives thereof, and the like.
- the targeting ligand may be derivatized to an appropriate portion of the cationic polymer prior to the formation of the polynucleotide delivery vehicle.
- the targeting agent e.g., immunoglobulin
- the targeting agent may be N- linked to a free carboxyl group of the polar region of a branched cross-linking molecule, by first derivatizing a leaving group to the carboxyl group using N—hydroxysuccinimide (NHS) and l-ethyl-3- (3- dimethylaminopropyl) carbodiimide (EDAC) , or the methiodide thereof, (EDC methiodide) and a free amino group on the targeting molecule.
- NHS N—hydroxysuccinimide
- EDAC l-ethyl-3- (3- dimethylaminopropyl) carbodiimide
- EDC methiodide methiodide
- targeting agents may be disulfide linked to a properly conditioned linking agent or cation (using thioacetic acid, hydroxylamine, and EDTA) .
- PDVs comprise lipids
- succinimidyl acetylthioacetate may be used in conjunction with a fatty acid (e.g., dioleylphosphatidyl-ethanolamine, DOPE) to form a DOPE-thioacetate (ATA) which may be treated with hydroxylamine to generate the reduced molecule (DOPE-acetyl- SH) .
- DOPE dioleylphosphatidyl-ethanolamine
- a free amino group on the targeting agent is reacted with succinimidyl maleimidophenyl butyrate (SMPB) to produce a target which is linked to maleimidophenylbutyrate (MPB) by a peptide bond.
- SMPB succinimidyl maleimidophenyl butyrate
- MPB maleimidophenylbutyrate
- the derivatized fatty acid is subsequently combined with the target-MPB complex to produce a targeting agent which has been cross-linked to a fatty acid.
- the targeting agent may be linked to the lipid by a biodegradable linkage as discussed above (peptide or dipeptide linkers, pH hydrolyzable linkers, etc.) .
- the targeting agent may also act as a bridge between the PDVs and the "targeted" cells or tissues.
- the agent may be added to the complex well after complex formation or isolation.
- the targeting agent may act as a bridge molecule which effectively places the complex in intimate contact with the cell surface.
- hepatocytes are the preferred target of PDV-directed transfection
- molecules such as fetuin may prove useful.
- Hepatocytes contain a galactose receptor. After treatment with neuraminidase, fetuin is converted to asialofetuin which displays a number of galactose residues on its surface.
- both fetuin and asialofetuin are known to associate with the DNA complexes comprising cationic lipids.
- asialofetuin As a molecule rich in acidic amino acids (aspartic acid and glutamate) asialofetuin (ASF) presumably associates the cationic groups of DNA/cation complexes. Consequently, asialofetuin-associated complexes are targeted to hepatocytes by virtue of the exposed galactose residues on the protein. The observation that asialofetuin associates with DNA/cation complexes also has far reaching potential. For instance, asialofetuin may be derivatized with any of a wide number to targeting ligands using any of a number of conventional chemical methods.
- periodate may be used to convert at least a portion of the hydroxyl groups on galactose to aldehydes, the aldehydes react with primary amino groups to form Schiff bases, which may be subsequently be reduced with lithium aluminum hydride (to add a targeting ligand) .
- the aldehydes may be reacted with hydrazide to attach heterobifunctional cross-linking reagents (which has been to suitable targeting ligands) .
- Either of the above strategies are simply illustrative of the many possible ways asialofetuin may be derivatized with practically any targeting ligand, and should not be construed as limiting the invention in any way.
- the derivatized asialofetuin may be associated with the DNA/cation complex as described above.
- Virtually any ligand can be attached to asialofetuin, and virtually any DNA can be packaged into the stable complex.
- virtually any cell may be targeted to express virtually any gene.
- asialofetuin, or functional equivalents thereof may be N-linked to the cationic polymer and directly incorporated into PDVs.
- proteins that associate with the PDVs may be suitably derivatized with a targeting ligand and used to direct PDVs to specific cells and tissues.
- any of a variety of cells such as endothelial cells, line cells, epithelial cells, islets, neurons or neural tissue, mesothelial cells, osteocytes, chondrocytes, hematopoietic cells, immune cells, cells of the major glands or organs (e.g., lung, heart, stomach, pancreas, kidney, skin, etc.), exocrine and/or endocrine cells, and the like, may be targeted for gene delivery.
- cells such as endothelial cells, line cells, epithelial cells, islets, neurons or neural tissue, mesothelial cells, osteocytes, chondrocytes, hematopoietic cells, immune cells, cells of the major glands or organs (e.g., lung, heart, stomach, pancreas, kidney, skin, etc.), exocrine and/or endocrine cells, and the like, may be targeted for gene delivery.
- major glands or organs e.g
- proteins encoding various cell surface markers and receptors.
- a brief list that is exemplary of such proteins includes: CDl(a-c), CD4, CD8-ll(a- c) , CD15, CDwl7, CD18, CD21-25, CD27, CD30-45(R(O, A, and B) ) , CD46-48, CDw49 (b,d, f) , CDw50, CD51, CD53-54, CDw60,
- polynucleotide delivery vehicles comprising the described cationic polymers and cationic lipids generally retain transfection efficiencies of at least about twenty (20) percent of the polynucleotide transfection efficiency of freshly prepared product after storage for forty-eight (48) hours, and preferably retain at least about thirty-five (35) percent transfection efficiency after 48 hours, and in a particularly preferred embodiment will retain at least about fifty (50) percent transfection efficiency after 48 hours.
- the presently described PDVs remain size- stable and generally retain a discrete size range of between about 50 and about 1,000 nm, preferably between about 75 and about 600 nm, and preferably between about 100 and about 450 nm average particle size (as per a Gaussian distribution) after being held in the liquid state for at least 48 hours.
- PDVs formed by ethanol evaporation are smaller (mean diameter less than about 150 nm) than PDVs formed by detergent dialysis (mean diameter greater than about 200 nm) .
- the presently described PDVs are preferably serum stable in that they are generally at least about twice as stable than, and preferably at least about one order of magnitude more stable than liposomal formulations produced using the methods/synthetic cationic lipids taught by the prior art when exposed to serum concentrations of up to about fifteen (15) percent.
- the stability of the presently described PDVs may be augmented by the appropriate storage conditions.
- the PDVs may be frozen and stored indefinitely. After rapid or slow (at about 4°C) thawing, the PDVs typically retain a substantial portion, if not all, of the transfection efficiency of freshly produced samples. Moreover, the subject PDVs also retain a substantial amount (i.e., at least about 50 percent) of their original transfection efficiency after lyophilization and reconstitution.
- suitable excipients may be added to the PDV preparation prior to freezing.
- suitable excipients include, mono or disaccharides (e.g., glucose, sucrose, etc.), polysaccharides, or any of a variety of well-known agents (e.g., glycerols, gums, dextrans,_ and the like) .
- PDVs may aggregate.
- a loose aggregate is defined as an aggregate that is easily dispersible into suspension.
- such aggregation may occur after a period of frozen storage (at about -20° C or less) , followed by thawing.
- the level of aggregation may be regulated by any of a number of means in addition to adjusting temperature.
- buffer/salt concentration may be adjusted to increase the amount of aggregation.
- coprecipitants may be added which complex with the stable complexes and further increase the rate of extent of precipitation. Aggregation may also be increased by the addition of facilitating agents.
- a suitable lectin, ligand, or antibody may be added to cross-link the complexes and increase the rate and extent of aggregation or precipitation.
- a suitable ligand or antibody, or mixture thereof may be affixed to a suitable solid support, i.e., latex beads, microcarrier beads, membranes or filters, and the like, and used to selectively bind PDVs which incorporate the targeting receptor or ligand from the preparation.
- a method is provided for isolating the desired PDVs prior to use.
- an additional embodiment of the present invention is a method of producing PDVs that retain measurable transfection activity and comprise at least about 10 ⁇ g of nucleic acid per ml up to about 10 mg/ml.
- another embodiment of the present invention is a method of producing PDVs of substantially reduced toxicity.
- the terms "substantially reduced toxicity” or “substantially nontoxic” shall mean that the toxicity of an agent shall generally be reduced by at least about 25 percent relative to existing cation-derivatized polymers (i.e., DEAE-dextran, and the like) , preferably by at least about 50 percent, and optimally a reduction of at least about a 100 percent will be achieved. Toxicity may also be measured by determining the dose which is lethal to fifty percent of the test subjects.
- the described PDVs will have a lethal dose, or LD 50 , twice that of nonisolated stable complex formed at similar cationic lipid/phosphate ratios, and optimally reduced toxicity vehicles will have an LD 50 at least about one order of magnitude greater than that of DEAE-dextran.
- any of a variety of stabilizing agents may be utilized in conjunction with the described vehicles.
- oxidation of the various components may be substantially reduced by preparing formulations in accordance with the present invention under an inert atmosphere, such as nitrogen, this is a somewhat inconvenient and expensive process and so it is often preferred to add chemical anti- oxidants.
- Suitable pharmaceutically acceptable antioxidants include propyl gallate, butylated hydroxyanisole, butylated hydroxytoluene, ascorbic acid or sodium ascorbate, DL- or D- alpha tocopherol and DL- or D-alpha-tocopheryl acetate.
- the anti-oxidant may be added singly or in combination to the polynucleotide delivery vehicles either before, during, or after vehicle assembly in an amount of up to, for example, 0.1% (w/v) , preferably from 0.0001 to 0.05%.
- an undesirable symptom e.g., symptoms related to disease, sensitivity to environmental or factors, normal aging, and the like.
- treatment shall refer to any and all uses of the claimed compositions which remedy a disease state or symptoms, or otherwise, prevent, hinder, retard, or reverse the progression of disease or other undesirable symptoms in any way whatsoever.
- an appropriate dosage of polynucleotide delivery vehicle (PDV) may be determined by any of several well established methodologies. For instance, animal studies are commonly used to determine the maximal tolerable dose, or MTD, of bioactive agent per kilogram weight. In general, at least one of the animal species tested is mammalian. Those skilled in the art regularly extrapolate doses for efficacy and avoiding toxicity to other species, including human. Before human studies of efficacy are undertaken, Phase I clinical studies in normal subjects help establish safe doses.
- the various biochemical components of the present invention are preferably of high purity and are substantially free of potentially harmful contaminants (e.g., at least National Food (NF) grade, generally at least analytical grade, and preferably at least pharmaceutical grade) .
- potentially harmful contaminants e.g., at least National Food (NF) grade, generally at least analytical grade, and preferably at least pharmaceutical grade
- NF National Food
- synthesis or subsequent purification shall preferably result in a product that is substantially free of any potentially toxic agents which may have been used during the synthesis or purification procedures.
- PDVs may also be modified to enhance in vivo stability as well as any of a variety of pharmacological properties (e.g., increase in vivo half-life, further reduce toxicity, etc.) by established methods. For instance, by varying the extent of cross-linking and branching in the cationic polymer, the physiological characteristics of the PDVs may be altered. This makes is possible to construct
- PDVs that are capable of delivering nucleic acid to the body in a time-released manner. Such time release formulations are contemplated to facilitate the treatment of acute conditions by providing extended periods of transient gene delivery,_ or providing practitioners with alternative means of dosaging and delivering nucleic acid in vivo . In particular, the presently described PDVs are ideal for the packaging and delivery of nucleic acid based vaccines.
- the PDV may be prepared and maintained under sterile conditions and thus avoid microbial contamination. Because of the relatively small size and inherent stability of the PDVs, compositions comprising PDVs may also be sterile filtered prior to use. In addition to the above methods of sterile preparation and filter sterilization, antimicrobial agents may also be added.
- Antimicrobial agents which may be used, generally in amounts of up to about 3% w/v, preferably from about 0.5 to 2.5%, of the total formulation, include, but are not limited to, methylparaben, ethylparaben, propylparaben, butylparaben, phenol, dehydroacetic acid, phenylethyl alcohol, sodium benzoate, sorbic acid, thymol, thimerosal, sodium dihydroacetate, benzyl alcohol, cresol, p-chloro-m-cresol, chlorobutanol, phenylmercuric acetate, phenylmercuric borate, phenylmercurie nitrate and benzylalkonium chloride.
- anti-microbial additives will either enhance the biochemical properties of the PDVs, or will be inert with respect to PDV activity.
- another agent may be substituted which effects PDV function to a lesser extent.
- compositions comprising PDVs as active components may be introduced in vivo by any of a number of established methods.
- the agent may be administered by inhalation; by subcutaneous (sub-q) ; intravenous (I.V.), intraperitoneal (I.P.), or intramuscular (I.M.) injection; rectally, as a topically applied agent (transdermal patch, ointments, creams, salves, eye drops, and the like) , or directly injected into tissue such as tumors or other organs, or in or around the viscera.
- a topically applied agent transdermal patch, ointments, creams, salves, eye drops, and the like
- Another embodiment of the subject invention involves the use of PDVs to effect gene therapy.
- Such gene therapy is intended to compensate for genetic deficiencies in the afflicted individual's genome and may be effected by ex vivo somatic cell gene therapy whereby host cells are removed from the body are transduced to express the deficient gene and reimplanted into the host.
- somatic cell gene therapy may be effected by directly injecting a vector bearing the desired gene into the individual, in vivo, whereby the gene will be delivered and expressed by host tissue.
- polynucleotide delivery vehicles are primarily intended to provide polynucleotides to cells
- a further embodiment of the present invention contemplates the packaging and delivery of any of a variety of suitable bioactive agents in addition to polynucleotides.
- a bioactive agent e.g., any protein, peptide, small organic molecule, and the like
- a given agent of interest may associate with polynucleotide (e.g., proteins or other molecules with DNA and/or RNA binding activity)
- polynucleotide e.g., proteins or other molecules with DNA and/or RNA binding activity
- stabilizers and/or plasticizers may be added to PDV formulations for greater storage stability.
- Materials useful as stabilizers and/or' plasticizers include simple carbohydrates including, but not limited to, glucose, galactose, sucrose, or lactose, dextrin, acacia, carboxypolymethylene and colloidal aluminum hydroxide.
- stabilizers/plasticizers When stabilizers/plasticizers are added, they may be incorporated in amounts up to about 10% (w/v) , preferably from about 0.5 to 6.5%, of the total preparation.
- Lipid formulations e.g., emulsions, microemulsions, liposomes, or delivery vehicles
- PDVs may also prove useful for the oral administration of bioactive agents.
- enteric protection it is possible to formulate solid or liquid formulations in accordance with the invention in an enteric-coated or otherwise protected form.
- solid formulations they can either be mixed or simply coadministered with a protectant, such as a liquid mixture of medium chain triglycerides, or they can be filled into enteric capsules (for example of soft or hard gelatin, which are themselves optionally additionally enteric coated.
- solid, or dry (i.e., desiccated or lyophilized) formulations of PDVs may be treated more flexibly.
- enteric materials may either be coated with enteric materials to form tablets or they can be filled into enteric capsules.
- the thickness of enteric coating on tablets or capsules can be, for example, from 0.5 to 4 microns in thickness, although the precise thickness will be determined by the skilled formulator.
- Enteric coated granules (whose particle size may be, for example, from 0.5 to 2mm) may themselves be coated without being compounded into a tablet for coating.
- Microcapsules similarly, can be enteric coated.
- the enteric coating may comprise any of the enteric materials conventionally utilized in orally administrable pharmaceutical formulations. Suitable enteric coating materials are known, for example, from "Remington's
- enteric coating materials examples include cellulose acetylphthalate, hydroxypropylmethylcellulose- phthalate (HPMC-P) , benzophenyl salicylate, cellulose acetosuccinate, copolymers of styrene and maleic acid, formulated gelatin, keratin, stearic acid, myristic acid, polyethylene glycol, shellac, gluten, acrylic and methacrylic resins and copolymers of maleic acid and phthalic acid derivatives.
- the enteric coating material (s) may be dissolved in solvents such as dichloromethane, ethanol and water, cellulose phthalate, or polyvinyl acetate phthalate.
- HPMC-P polyethylene glycol 6000 or shellac
- HP5-5 a proprietary preparation of HPMC-P aimed at dissolution or dissipation at pH 5.5, which is encountered in the human pyrolus, is available under the trade mark HP5-5, and is particularly preferred.
- the presently disclosed cationic polymers, and polynucleotide delivery vehicles produced therewith, represent a marked improvement over currently available synthetic cationic lipids vis-a-vis polynucleotide delivery to cells because the byproducts of the degradation reaction are substantially nontoxic, or inherently biocompatible.
- the presently disclosed cationic polymers are be useful for the delivery of polynucleotides to cells in vi tro as well as in vivo.
- biocompatible pH sensitive or otherwise biodegradable linker portion of the cationic polymer to attach other biocompatible or groups in place of the presently disclosed cationic groups.
- bioactive molecules may be functionally derivatized to polymers as described above and delivered to the body in a controlled release manner.
- proteinaceous biological material examples include, but are not limited to, protein hormones such as insulin, calcitonin and growth hormone, whether from human or animals or semi- or totally synthetically prepared, erythropoietin, plasminogen activators and their precursors, such as tPA, urokinase, pro- urokinase and streptokinase, interferons including human interferon alpha, interleukins including IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7 and IL-12, and blood factors including Factor VIII.
- protein hormones such as insulin, calcitonin and growth hormone, whether from human or animals or semi- or totally synthetically prepared
- erythropoietin plasminogen activators and their precursors
- tPA urokinase
- pro- urokinase and streptokinase interferons including human interferon alpha
- interleukins
- Figure 1 provides an overview of a method for producing cationic polymers.
- the polycation PEHA is specifically shown but it is contemplated that similar cations comprising lesser, greater, or more highly branched amine groups are equally useful.
- the linking group shown in Figure 1 is a dicarboxylic acid that cross-links the PEHA monomers by amide linkages.
- Figure 2 provides a description of a fraction of the various other cationic groups, cross-linking agents, functional groups, and branched cross-linking agents, that may be also be used to construct cationic polymers for gene delivery.
- alternate biogenic amines are shown.
- homofunctional cross-linking agents iminothiolane, dithioJbis(succinimidylpropionate) , and disuccinimidyltartarate are shown.
- the molecule N-BOC- glutamic acid is provided as an example of how additional functional groups may be incorporated into the cationic polymer (using carboxylic acid groups on the amino acid) , and the use of citric acid and ethylenediaminetetraacetic acid (EDTA) as branched cross-linking agents is also shown.
- EDTA ethylenediaminetetraacetic acid
- Reagent grade PEHA was obtained from Aldrich chemicals and analysis showed that the molecule, as provided, was about eighty percent full-length product mixed with a variety of shorter, and longer, synthesis products. Where in vivo use is contemplated, all reagents will be of the highest purity available, and preferably of pharmaceutical grade or better.
- PEHA is polymerized using cross-linker by slowly adding PEHA to an excess of linker (with stirring at room about room temperature) . During the reaction, the resulting polymer may precipitate from solution and facilitate isolation of the product. Alternatively, the relative concentrations of the reagents may be reversed. By varying the duration of the polymerization reaction or the reaction conditions, one can produce polymers comprising a wide range of average molecular weights . Under the specified conditions, PEHA polymers with a mean molecular weight between about 100,000 to about 400,000 daltons are produced.
- PEHA polymer was hydrated in a suitable buffer (for example, 150 mM NaCl; 50 mM NaCl; 10 mM NaCl; 0.2 M dextrose; 50 mM NaCl, 0.2 M dextrose; or 150 mM NaCl, 0.2 M dextrose) at a concentration of about 450 /xg of polymer/ml, or up to about 4 mg per ml.
- DNA (SSV9-pMD-AP) was added to the PEHA polymer at cation/DNA phosphate ratio of between about 1:1 and about 20:1, and incubated for about 10 to 30 minutes.
- concentration and type of salt present during complex formation will vary dependent upon the intended use of the complex (i.e., in vi tro versus in vivo) .
- the resulting cation/DNA complex was either directly applied to cells or is injected into mouse tail veins (I.V.) as a composition comprising about 60 ⁇ g of DNA in about 300 ⁇ l . 6.2.2. Protocol for Formulating DNA/Cationic Lipid Complexes bv Alcohol Evaporation.
- DOSPA lipofectamine
- a DNA solution was prepared at a concentration of 1.2 mg/ml in 40 percent ethanol/250 mM glucose/25 mM NaCl. An equal volume of DNA was then added to the lipid solution to yield a final DNA concentration of about .63 mg/ml and a final DOSPA/DNA nucleotide ratio of about 0.6.
- the ethanol/water solution was removed by rotoevaporation which resulted in a thin dry film of DNA/cation complex. The film was then hydrated with water to yield a stable solution of PDVs.
- Figure 5 shows in vivo expression data obtained using the PDVs, and, inter alia , compares results obtained using PDVs prepared in the presence or absence of NaCl .
- TAC was used in lieu of DOSPA, it was used at a TAC:DOPE (mol/mol) ratio of about 75:25, the complexation buffer preferably had a pH of about 6, and the complexation reaction preferably occured at about room temperature.
- Particle size analysis was obtained using a Leeds and Northrop laser dynamic light scattering instrument. Characterization of the cationic polymer (in water) showed that particles were formed with a mean size of approximately 200 nm in diameter. The addition of NaCl (150 mM) caused the mean size to increase to about 1,000 nm. The addition of DNA caused the mean size of the particles to decrease to about 400 nm.
- PDVs prepared using the alcohol evaporation method typically have a mean diameter of less than 200 nm, and may be extruded to form particles of less than 100 nm mean diameter.
- PDVs prepared using TAC may be extruded to yield a mean particle size of between about 40 and about 100 nm.
- PDVs were formed essentially as described in section 6.2 (150 mM NaCl) and added to approximately 10 s NIH 3T3 cells cultured in 0.5 ml of serum free media. After the PDVs (about 10 ⁇ g of DNA) were added, the cells were incubated for about 4 hr. The cells were subsequently assayed for expression of the reporter gene by an alkaline phosphatase immunocapture assay. These studies revealed that the PDVs are useful for gene delivery in vi tro .
- PDVs formed essentially as described in section 6.2 were injected into mice as follows. PDVs comprising approximately 60 ⁇ g of DNA were injected into mouse tail veins in a net volume of about 500 ⁇ l . Mouse tissue samples were harvested 48 hours after PDV administration and homogenized in buffer at a net concentration of about 100 mg/ml .
- the homogenates were heated to 65° C for 30 minutes to inactivate endogenous alkaline phosphatase, and analyzed using an immunocapture assay 'comprised of adsorbing a secondary antibody to a 96 well plate that binds a subsequently added anti-human placental alkaline phosphatase polyclonal antibody.
- 0.2 ml of homogenate was added to each well and allowed to incubate overnight at 4° C. The wells were washed, additional 200 ⁇ l aliquots were added to the wells and incubated for 2 hours to increase the signal, the wells were washed again, and an alkaline phosphatase substrate was added.
- the plate was then read using a Molecular Devices plate reader which can determine a V m for each well.
- the V ⁇ was converted to mUnits of AP and the data were normalized per 100 mg of tissue using a standard curve ranging from 20 mUnits to 0.1 mUnits of alkaline phosphatase (AP) .
- AP alkaline phosphatase
- cardiopulmonary tissues tend to best express genes delivered by PDVs formed by the detergent dialysis method, and delivered as described.
- the extent and areas of expression may correspondingly vary.
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Abstract
Description
Claims
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JP54298297A JP2001508815A (en) | 1996-05-29 | 1997-05-29 | Cationic polymer / lipid nucleic acid delivery vehicle |
AU32909/97A AU739969B2 (en) | 1996-05-29 | 1997-05-29 | Cationic polymer/lipid nucleic acid delivery vehicles |
EP97928725A EP0904031A4 (en) | 1996-05-29 | 1997-05-29 | Cationic polymer/lipid nucleic acid delivery vehicles |
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US1837796P | 1996-05-29 | 1996-05-29 | |
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PCT/US1997/009275 WO1997045069A1 (en) | 1996-05-29 | 1997-05-29 | Cationic polymer/lipid nucleic acid delivery vehicles |
Country Status (6)
Country | Link |
---|---|
US (2) | US20010007771A1 (en) |
EP (1) | EP0904031A4 (en) |
JP (1) | JP2001508815A (en) |
AU (1) | AU739969B2 (en) |
CA (1) | CA2256900A1 (en) |
WO (1) | WO1997045069A1 (en) |
Cited By (12)
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WO1998019710A2 (en) * | 1996-11-06 | 1998-05-14 | Etienne Honore Schacht | Delivery of nucleic acid material to target cells in biological systems |
WO1998029541A1 (en) * | 1997-01-03 | 1998-07-09 | Mirus Corporation | A process of making a compound by forming a polymer from a template drug |
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WO1999058151A2 (en) * | 1998-05-07 | 1999-11-18 | Universiteit Gent | Synthetic polyaminoacid complexes for delivery of nucleic acids to target cells |
WO2003097107A2 (en) * | 2002-05-14 | 2003-11-27 | Nitto Denko Corporation | Controllably degradable polymeric biomolecule or drug carrier and method of synthesizing said carrier |
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5629184A (en) * | 1995-01-25 | 1997-05-13 | Amgen Inc. | Cationic copolymers of vinylamine and vinyl alcohol for the delivery of oligonucleotides |
US5635380A (en) * | 1994-01-18 | 1997-06-03 | Vanderbilt University | Enhancement of nucleic acid transfer by coupling virus to nucleic acid via lipids |
US5656611A (en) * | 1994-11-18 | 1997-08-12 | Supratek Pharma Inc. | Polynucleotide compositions |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB8913737D0 (en) * | 1989-06-15 | 1989-08-02 | Univ Birmingham | A novel anti-allergy treatment |
DE4130786A1 (en) * | 1991-09-16 | 1993-03-18 | Symbiotec Gmbh | PEPTIDES FOR PRODUCING AGENTS FOR DIAGNOSIS AND THERAPY OF SYSTEMIC LUPUS |
FR2715847B1 (en) * | 1994-02-08 | 1996-04-12 | Rhone Poulenc Rorer Sa | Composition containing nucleic acids, preparation and uses. |
FR2719316B1 (en) * | 1994-04-28 | 1996-05-31 | Idm | New nucleic acid and polymer complexes, their preparation process and their use for cell transfection. |
US5948878A (en) * | 1997-04-15 | 1999-09-07 | Burgess; Stephen W. | Cationic polymers for nucleic acid transfection and bioactive agent delivery |
-
1997
- 1997-05-29 EP EP97928725A patent/EP0904031A4/en not_active Withdrawn
- 1997-05-29 CA CA002256900A patent/CA2256900A1/en not_active Abandoned
- 1997-05-29 US US08/865,375 patent/US20010007771A1/en not_active Abandoned
- 1997-05-29 AU AU32909/97A patent/AU739969B2/en not_active Ceased
- 1997-05-29 WO PCT/US1997/009275 patent/WO1997045069A1/en not_active Application Discontinuation
- 1997-05-29 JP JP54298297A patent/JP2001508815A/en active Pending
-
2002
- 2002-02-28 US US10/084,159 patent/US20020082237A1/en not_active Abandoned
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5635380A (en) * | 1994-01-18 | 1997-06-03 | Vanderbilt University | Enhancement of nucleic acid transfer by coupling virus to nucleic acid via lipids |
US5656611A (en) * | 1994-11-18 | 1997-08-12 | Supratek Pharma Inc. | Polynucleotide compositions |
US5629184A (en) * | 1995-01-25 | 1997-05-13 | Amgen Inc. | Cationic copolymers of vinylamine and vinyl alcohol for the delivery of oligonucleotides |
Non-Patent Citations (1)
Title |
---|
See also references of EP0904031A4 * |
Cited By (25)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998019710A2 (en) * | 1996-11-06 | 1998-05-14 | Etienne Honore Schacht | Delivery of nucleic acid material to target cells in biological systems |
WO1998019710A3 (en) * | 1996-11-06 | 1998-09-17 | Etienne Honore Schacht | Delivery of nucleic acid material to target cells in biological systems |
WO1998029541A1 (en) * | 1997-01-03 | 1998-07-09 | Mirus Corporation | A process of making a compound by forming a polymer from a template drug |
US7531676B2 (en) | 1998-04-08 | 2009-05-12 | Celltech R & D Limited | Lipids |
AU768542C (en) * | 1998-04-08 | 2004-06-17 | Celltech R & D Limited | Lipids |
WO1999052858A2 (en) * | 1998-04-08 | 1999-10-21 | Celltech Therapeutics Limited | Lipids |
AU768542B2 (en) * | 1998-04-08 | 2003-12-18 | Celltech R & D Limited | Lipids |
WO1999052858A3 (en) * | 1998-04-08 | 2000-04-06 | Celltech Therapeutics Ltd | Lipids |
US6583301B1 (en) | 1998-04-08 | 2003-06-24 | Celltech R & D Limited | Lipids |
JP2002511446A (en) * | 1998-04-08 | 2002-04-16 | セルテック・セラピューティクス・リミテッド | Fat |
EP0963758A3 (en) * | 1998-05-07 | 2000-03-22 | Universiteit Gent | Synthetic polyaminoacid complexes for delivery of nucleic acids to target cells |
WO1999058151A2 (en) * | 1998-05-07 | 1999-11-18 | Universiteit Gent | Synthetic polyaminoacid complexes for delivery of nucleic acids to target cells |
WO1999058151A3 (en) * | 1998-05-07 | 2000-04-13 | Univ Gent | Synthetic polyaminoacid complexes for delivery of nucleic acids to target cells |
EP0963758A2 (en) * | 1998-05-07 | 1999-12-15 | Universiteit Gent | Synthetic polyaminoacid complexes for delivery of nucleic acids to target cells |
WO2003097107A2 (en) * | 2002-05-14 | 2003-11-27 | Nitto Denko Corporation | Controllably degradable polymeric biomolecule or drug carrier and method of synthesizing said carrier |
WO2003097107A3 (en) * | 2002-05-14 | 2004-01-22 | Nitto Denko Corp | Controllably degradable polymeric biomolecule or drug carrier and method of synthesizing said carrier |
US8192989B2 (en) | 2003-01-13 | 2012-06-05 | Nitto Denko Corporation | Solid surface for biomolecule delivery and high-throughput assay |
US7163677B2 (en) | 2003-10-24 | 2007-01-16 | Nitto Denko Corporation | Cationic polymers having degradable crosslinks |
US7358223B2 (en) | 2004-10-04 | 2008-04-15 | Nitto Denko Corporation | Biodegradable cationic polymers |
US8258235B2 (en) | 2004-10-04 | 2012-09-04 | Nitto Denko Corporation | Biodegradable cationic polymers |
US7700542B2 (en) | 2004-10-04 | 2010-04-20 | Nitto Denko Corporation | Biodegradable cationic polymers |
WO2007020060A1 (en) * | 2005-08-17 | 2007-02-22 | Universität Regensburg | Cationic polymer for transporting nucleic acids in cells |
US7700541B2 (en) | 2006-04-06 | 2010-04-20 | Nitto Denko Corporation | Biodegradable cationic polymers |
WO2013055865A1 (en) | 2011-10-11 | 2013-04-18 | The Brigham And Women's Hospital, Inc. | Micrornas in neurodegenerative disorders |
WO2018081817A2 (en) | 2016-10-31 | 2018-05-03 | University Of Massachusetts | Targeting microrna-101-3p in cancer therapy |
Also Published As
Publication number | Publication date |
---|---|
AU3290997A (en) | 1998-01-05 |
CA2256900A1 (en) | 1997-12-04 |
JP2001508815A (en) | 2001-07-03 |
EP0904031A4 (en) | 2002-05-02 |
AU739969B2 (en) | 2001-10-25 |
US20010007771A1 (en) | 2001-07-12 |
EP0904031A1 (en) | 1999-03-31 |
US20020082237A1 (en) | 2002-06-27 |
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