EP1100889A1 - Solutions de tensio-actifs - Google Patents

Solutions de tensio-actifs

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Publication number
EP1100889A1
EP1100889A1 EP99935624A EP99935624A EP1100889A1 EP 1100889 A1 EP1100889 A1 EP 1100889A1 EP 99935624 A EP99935624 A EP 99935624A EP 99935624 A EP99935624 A EP 99935624A EP 1100889 A1 EP1100889 A1 EP 1100889A1
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EP
European Patent Office
Prior art keywords
nucleic acid
cells
complex
dna
cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP99935624A
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German (de)
English (en)
Other versions
EP1100889A4 (fr
Inventor
Jon A. Wolff
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Arrowhead Madison Inc
Original Assignee
Mirus Corp
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Filing date
Publication date
Application filed by Mirus Corp filed Critical Mirus Corp
Publication of EP1100889A1 publication Critical patent/EP1100889A1/fr
Publication of EP1100889A4 publication Critical patent/EP1100889A4/fr
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Definitions

  • the invention generally relates to micellar systems for use in biologic systems. More particularly, a process is provided for the use of reverse micelles for the delivery of nucleic acids and genes to cells.
  • Biologically active compounds such as proteins, enzymes, and nucleic acids have been delivered to the cells using amphipathic compounds that contain both hydrophobic and hydrophilic domains.
  • amphipathic compounds are organized into vesicular structures such as liposomes, micellar, or inverse micellar structures.
  • Liposomes can contain an aqueous volume that is entirely enclosed by a membrane composed of lipid molecules (usually phospholipids) (R.C. New, p. 1, chapter 1, "Introduction” in Liposomes: A Practical Approach, ed. R.C. New IRL Press at Oxford University Press, Oxford, 1990).
  • Micelles and inverse micelles are microscopic vesicles that contain amphipathic molecules but do not contain an aqueous volume that is entirely enclosed by a membrane. In micelles the hydrophilic part of the amphipathic compound is on the outside (on the surface of the vesicle) whereas in inverse micelles the hydrophobic part of the amphipathic compound is on the outside.
  • the inverse micelles thus contain a polar core that can solubilize both water and macromolecules within the inverse micelle.
  • a polar core that can solubilize both water and macromolecules within the inverse micelle.
  • the resulting inverse micelle can be referred to as a microemulsion of water in oil (Schelly, Z. A. Current Opinion in Colloid and Interface Science, 37-41, 1997; Castro, M J. M., Cabral, J. M. S. Biotech. Adv. 6, 151-167, 1988).
  • Microemulsions are isotropic, thermodynamically stable solutions in which substantial amounts of two immicible liquids (water and oil) are brought into a single phase due to a surfactant or mixture of surfactants.
  • the spontaneously formed colloidal particles are globular droplets of the minor solvent, surrounded by a monolayer of surfactant molecules.
  • the spontaneous curvature, Ho of the surfactant monolayer at the oil/water interface dictates the phase behavior and microstructure of the vesicle.
  • Hydrophilic surfactants produce oil in water (O/W) microemulsions (Ho>0), whereas lipophilic surfactants produce water in oil (W/O) microemulsions.
  • Ho bicontinuous-type microemulsions are formed
  • Positively-charged, neutral, and negatively-charged liposomes have been used to deliver nucleic acids to cells.
  • plasmid DNA expression in the liver has been achieved via liposomes delivered by tail vein or intraportal routes.
  • Positively- charged micelles have also been used to package nucleic acids into complexes for the delivery of the nucleic acid to cells.
  • Negatively-charged micelles have been used to condense DNA, however they have not been used for the delivery of nucleic acids to cells (Imre, V. E., Luisi, P. L. Biochemical and Biophysical Research Communications, 107, 538-545, 1982).
  • DNA amphipathic complexes including micelles in which at least one amphipathic compound layer that surrounds a non-aqueous core that contains a polyion such as a nucleic acid (Wolff, J., Budker, V., and Gurevich, V. U.S. Patent # 5635487).
  • Cleavable Micelles in which at least one amphipathic compound layer that surrounds a non-aqueous core that contains a polyion such as a nucleic acid (Wolff, J., Budker, V., and Gurevich, V. U.S. Patent # 5635487).
  • a new area in micelle technology involves the use of cleavable surfactants to form the micelle.
  • Surfactants containing an acetal linkage, azo-containing surfactants, elimination of an ammonium salt, quaternary hydrazonium surfactants, 2-alkoxy-N,N- dimethylamine N-oxides, and ester containing surfactants such as ester containing quaternary ammonium compounds and esters containing a sugar have been developed.
  • cleavable surfactants within micelles are designed to decompose on exposure to strong acid, ultraviolet light, alkali, and heat. These conditions are very harsh and are not compatible with retention of biologic activity of biologic compounds such as proteins or nucleic acids. Thus, biologically active compounds have not been purified using reverse micelles containing cleavable surfactants.
  • Polymers are used for drug delivery for a variety of therapeutic purposes. Polymers have also been used for the delivery of nucleic acids (polynucleotides and oligonucleotides) to cells for therapeutic purposes that have been termed gene therapy or anti-sense therapy.
  • nucleic acid delivery to the cells is the use of DNA-polycation complexes. It was shown that canonic proteins like histones and protamines or synthetic polymers like polylysine, polyarginine, polyornithine, DEAE dextran, polybrene, and polyethylenimine were effective intracellular delivery agents while small polycations like spermine were ineffective.
  • DNA translocation to the intracellular space might be non-specific adsorptive endocytosis which may be more effective then liquid endocytosis or receptor-mediated endocytosis.
  • polycations are a very convenient linker for attaching specific receptors to DNA and as result, DNA- polycation complexes can be targeted to specific cell types.
  • gene transfer enhancing signals that can be covalently attached to the gene in order to enable or enhance its cellular transport. These include signals that enhance cellular binding to receptors, cytoplasmic transport to the nucleus and nuclear entry or release from endosomes or other intracellular vesicles.
  • Nuclear localizing signals enhance the entry of the gene into the nucleus or directs the gene into the proximity of the nucleus.
  • Such nuclear transport signals can be a protein or a peptide such as the SV40 large T ag NLS or the nucleoplasmin NLS.
  • Other molecules include ligands that bind to cellular receptors on the membrane surface increasing contact of the gene with the cell. These can include targeting group such as agents that target to the asialoglycoprotein receptor by using asiologlycoproteins or galactose residues. Other proteins such as insulin, EGF, or transferrin can be used for targeting. Peptides that include the RGD sequence can be used to target many cells. Chemical groups that react with sulfhydryl or disulfide groups on cells can also be used to target many types of cells. Folate and other vitamins can also be used for targeting. Other targeting groups include molecules that interact with membranes such as fatty acids, cholesterol, dansyl compounds, and amphotericin derivatives.
  • Size of a DNA complex may be a factor for gene delivery in vivo. Many times, the size of DNA that is of interest is large, and one method of delivery utilizes compaction techniques.
  • the DNA complex needs to cross the endothelial barrier and reach the parenchymal cells of interest.
  • the largest endothelia fenestrae holes in the endothelial barrier
  • the trans-epithelial pores in other organs are much smaller, for example, muscle endothelium can be described as a structure which has a large number of small pores with a radius of 4 nm, and a very low number of large pores with a radius of 20-30 nm.(Rippe, B. Physiological Rev, 1994).
  • the size of the DNA complex is also important for the cellular uptake process. After binding to the target cells the DNA complex should be taken up by endocytosis. Since the endocytic vesicles have a homogenous internal diameter of about 100 nm in hepatocytes, and are of similar size in other cell types, the DNA is compacted to be smaller than 100 nm. Compaction of DNA
  • Multivalent cations with a charge of three or higher have been shown to condense DNA. These include spermidine, spermine, Co(NH 3 ) 6 3+ ,Fe 3+ , and natural or synthetic polymers such as histone HI, protamine, polylysine, and polyethylenimine.
  • DNA condensation has shown DNA condensation to be favored when 90% or more of the charges along the sugar-phosphate backbone are neutralized (Wilson, R. W., Bloomfield, V. A. Biochemistry 18, 2192-2196, 1979).
  • Polymers neutral or anionic which can increase repulsion between DNA and its surroundings have been shown to compact DNA.
  • Reverse micelles water in oil microemulsions
  • Reverse micelles water in oil microemulsions
  • examples exist of both recovery of extracellular proteins from a culture broth and recovery of intracellular proteins.
  • recovery of the biomolecules is difficult due to the stability of the formed micelle and due to incomplete recovery during the extraction process.
  • purification of DNA or other biomolecules from endotoxin and plasma is difficult to accomplish.
  • Triton results in incomplete separation of the DNA or biomolecules from the emulsion.
  • Reverse micelles have been widely used as a host for enzymatic reactions to take place.
  • enzymatic activity has been shown to increase with the used in a micelle, and has allowed enzymatic reactions to be conducted on water insoluble substrates.
  • enzymatic activity of whole cells entrapped in reverse micelles has been investigated (Gajjar, L., Singh, A., Dubey, R. S., Srivastava, R C. Applied Biochemistry and Biotechnology, 66, 159-172, 1997).
  • the cationic surfactant cetyl pyridinuim chloride was utilized to entrap Baker's yeast and Brewer's yeast inside a reverse micelle.
  • Micelles have also been used as a reaction media For example, a micelle has been used to study the kinetic and synthetic applications of the dehydrobromination of 2-(p- nitrophenyl)ethyl bromide. Additionally, micelles have found use as an emulsifier for emulsion polymerizations.
  • Micelles have been utilized for drug delivery.
  • an AB block copolymer has been investigated for the micellar delivery of hydrophobic drugs.
  • Transport and metabolism of thymidine analogues has been investigated via intestinal absorption utilizing a micellar solution of sodium glycocholate.
  • several examples of micelle use in transdermal applications have appeared.
  • sucrose laurate has been utilized for topical preparations of cyclosporin A.
  • the present invention provides for the transfer of polynucleotides, and biologically active compounds into parenchymal cells within tissues in situ and in vivo, utilizing reverse micelles delivered intravasculary, intrarterially, intravenous, orally, intraduodenaly, via the jejunum (or ileum or colon), rectally, transdermally, subcutaneously, intramuscularly, intraperitoneally, intraparenterally, via direct injections into tissues such as the liver, lung, heart, muscle, spleen, pancreas, brain (including intraventricular), spinal cord, ganglion, lymph nodes, lymphatic system, adipose tissues, thyroid tissue, adrenal glands, kidneys, prostate, blood cells, bone marrow cells, cancer cells, tumors, eye retina, via the bile duct, or via mucosal membranes such as in the mouth, nose, throat, vagina or rectum or into ducts of the salivary or other exocrine glands.
  • reverse micelles delivered intravasculary, intra
  • the polynucleotide becomes associated with the cell.
  • the polynucleotide can be on the membrane of the cell or inside the cytoplasm, nucleus, or other organelle of the cell.
  • the process of delivering a polynucleotide to a cell has also been commonly termed “transfection” or the process of "transfecting” and also it has been termed “transformation”.
  • the polynucleotide could be used to produce a change in a cell that can be therapeutic.
  • the delivery of polynucleotides or genetic material for therapeutic purposes is commonly called “gene therapy”.
  • the polynucleotides or genetic material being delivered are generally mixed with transfection reagents prior to delivery.
  • a biologically active compound is a compound having the potential to react with biological components.
  • Pharmaceuticals, proteins, peptides, hormones, cytokines, antigens and nucleic acids are examples of biologically active compounds.
  • the reverse micelle may be negatively-charged, zwitterionic, or neutral.
  • the present invention provides for the purification of biomolecules by solubilizing the biomolecule into a cleavable reverse micelle and then cleaving the micelle under conditions that will not destroy the biological activity of the biomolecule. These processes can be used for transferring nucleic acids or biomolecules into cells or an organism such as for drug delivery, or may also be used for analytical methods.
  • the process of utilizing cleavable reverse micelles for the purification of biomolecules has advantages over current methodology. Isolation of the biomolecule will be enhanced by cleaving the reverse micelle. This will separate the polar group from the non-polar group on the surfactant and therefore eliminate the formation of emulsions and therefore simplify the isolation process. Cleavage conditions will be such that the biological activity of the biomolecule is not destroyed.
  • the reverse micelle can compact the polynucleotide, a critical step for gene delivery, especially in vivo.
  • the micelle containing the compacted polynucleotide can then be utilized as a reaction vesicle in which additional compounds can be added to the DNA.
  • additional compounds can be added to the DNA.
  • a polycation could be added to the polynucleotide/reverse micelle solution to form a polycation/polynucleotide complex within the reverse micelle.
  • the polynucleotide/reverse micelle system is used as a vesicle for template polymerization of the DNA or caging of the DNA in which the polycation is crosslinked.
  • the polynucleotide / reverse micelle system also has advantages in that the micelle may be cleaved under physiological conditions involved along the "transfection pathway.”
  • the surfactant can be altered so that micellular cleavage occurs at different point along this pathway.
  • transfection pathway we mean any point at which the polynucleotide/reverse micelle system is introduced to a solution (i.e., blood, serum) that contains parenchymal cells, or to the cells (for example the skin or mucousal membranes) through the inclusion of the polynucleotide into the nucleus of the parenchymal cell.
  • a complex for delivery to a cell comprising: inserting a nucleic acid into a reverse micelle.
  • a compacting agent may be added to the complex as well as a delivery enhancing ligand or compound.
  • a process for delivering a complex to a cell comprising inserting a nucleic acid into a reverse micelle.
  • the nucleic acid or biomolecule is solubilized into a reverse micelle with an internal water volume for delivery of the biomolecule to parenchymal cells.
  • a compound can be added to the nucleic acid/micelle mixture.
  • Such compounds include polymers such as polyions (polycations such as spermine, polyamines, polylysine, polyethylimine (PEI), and polyanions), proteins, peptides, enzymes, hydrophobic compounds, and amphipathic compounds (to form a second layer around the micelle).
  • Such compounds include compounds that compact the DNA, provide a cell transfer enhancing ligand or provide another layer to the micelle.
  • the nucleic acid or biomolecule is complexed with another molecule prior to micelle formation.
  • a polymer such as polylysine is added to the nucleic acid and then crosslinked to cage the nucleic acid.
  • a polymer is formed since the polylysine (or any type of polymer) acts as a monomer that is being included in another polymer.
  • Another preferred embodiment provides a method of making a compound for delivery to a cell, comprising: adding one or more compounds to the nucleic acid or biomolecule/reverse micelle complex prior to delivery to the cell, thereby providing a deliverable complex.
  • a deliverable complex For example, another surfactant or a polyion might be added to the complex.
  • the cell can be a prokaryote or eukaryote and can be a plant, animal or mammalian cell.
  • Another preferred embodiment provides a method of making a compound for delivery to a cell, comprising: modifying a molecule in the presence of the biomolecule/reverse micelle complex thereby providing a deliverable complex.
  • the parenchymal cell is solubilized within a reverse micelle.
  • a reverse micelle containing a polynucleotide would be added to the parenchymal cell containing reverse micelle. After an appropriate amount of time, the parenchymal cell would be purified, and delivered to a mammal.
  • the biomolecule is solubilized utilizing one of the following procedures.
  • Procedure 1. a) mixing of the biomolecule into an aqueous solution b) then mixing the aqueous solution containing the biomolecule with a hydrocarbon or halohydrocarbon containing a surfactant with agitation or sonication
  • Procedure 2. a) mixing of the biomolecule into an aqueous solution b) then mixing the aqueous solution containing the biomolecule with a hydrocarbon or halohydrocarbon containing a surfactant with agitation or sonication Procedure 2.
  • the biomolecule is purified comprising a step in which a reverse micelle is destroyed.
  • the biomolecule can be purified by utilizing one of the following procedures.
  • Procedure 1. a) mixing of the biomolecule into an aqueous solution b) then mixing the aqueous solution containing the biomolecule with a hydrocarbon or halohydrocarbon containing a surfactant with agitation or sonication c) cleaving the reverse micelle d) extract the biomolecule Procedure 2.
  • the surfactant is a disulfide of general formula A-S- S-B, which contains a hydrophobic group (A) and a hydrophilic group (B).
  • the surfactant can be cleaved in the presence of the nucleic acid or biomolecule under conditions that will not destroy the biological activity of the nucleic acid or biomolecule.
  • the surfactant could be chemically modified in the presence of the nucleic acid or biomolecule.
  • the surfactant can be polymerized after micelle formation to form a "shell" or cage around the nucleic acid.
  • the surfactant could be cleaved separating the bulk of its hydrophilic and hydrophobic parts thus destroying its ability to act as a surfactant.
  • the possible surfactants can be neutral, negatively charged, or zwitterionic but not positively charged.
  • Neutral surfactants include (but not restricted to) polyoxyethylene alcohol's, polyoxyethylene isoalcohol, polyoxyethylene p-t-octyl phenol (Triton), polyoxyethylene nonylphenol, polyoxyehtylene esters of fatty acids, polyoxyethylene sorbitol esters (Tween) and lipids.
  • Negatively charged surfactants include (but not restricted to) di-(2-ethyl-hexyl) sodium sulfosuccinate (AOT), sodium dodecylsuphate (SDS), sodium dodecylsuphonate , and sodium dodecyl-N-sarcosinate.
  • the zwitterionic surfactant could contain anionic and cationic groups on the alpha and omega positions of a long aliphatic chain.
  • complex formation should be done under acidic conditions so that the surfactant can have a positive charge that will interact with the nucleic acid.
  • the anionic portion is neutralized by being protonated and therefore interacts with the non-aqueous phase.
  • the complexes are extracted into an aqueous solution containing a higher pH than the pH used to form the complexes.
  • a biologically active compound is purified is when it is isolated from a mixture of other compounds. It is purified when its purity is increased where purity is defined as the percentage of the mixture containing the desired biologically active compound. Purification can also indicate a process where the purity of the desired compound is not increased but the other compounds within the mixture are changed. Purification also indicates the extraction of a biologic compound from one mixture or solution to another mixture or solution. This can include a process where the desired biologically active compound is moved from one solvent to another (also called extraction) or is solubilized within a solvent.
  • a biologically active compound is a compound having the potential to react with biological components for gene therapy purposes.
  • a surfactant refers to a compound that contains a polar group (hydrophilic) and a non- polar (hydrophobic) group on the same molecule.
  • Cleavable surfactant refers to a surfactant in which the polar group may be separated from the nonpolar group by the removal of a chemical bond located between the two groups, or to a surfactant in which the polar or non-polar group or both may be chemically modified such that the detergent properties of the surfactant are destroyed.
  • Cleavable also means that the surfactant is labile (able to be destroyed or that the detergent properties of the surfactant are able to be destroyed) and that its surfactant properties could be destroyed by other chemical processes than bond cleavage.
  • a disulfide bond that is labile under physiological conditions means the disulfide bond is cleaved more rapidly than oxidized glutathione or any disulfide constructed from thiols in which one of the constituent thiols is more acidic, lower pKa, than glutathione or is activated by intramolecular attack by a free thiol. Constituent in this case means the thiols that are bonded together in the disulfide bond.
  • the surfactant properties of a compound can be destroyed by chemical modification such as converting the polar group into a less polar group.
  • chemical modification such as converting the polar group into a less polar group.
  • This can be accomplished by a number of chemical modifications including (but not limited to) acylation, alkylation, elimination, reduction or oxidation, of an amine (or its salt), alcohol, diol (di-alcohol) or carboxylic acid groups, or by a multistep process in which several chemical modifications are conducted (for example oxidation of an alcohol to an aldehyde (ketone) followed by nucleophilic addition to the aldehyde (ketone) resulting in an alcohol followed by elimination of the alcohol (or a derivative of it).
  • Reverse (inverse) micelle refers to a surfactant with an internal aqueous pool.
  • aqueous we mean containing water, but can include buffers and salts.
  • Non-aqueous solutions are made up of organic solvents such as hydrocarbon or halohydrocarbon.
  • Buffers are made from a weak acid or weak base and their salts. Buffer solutions resist changes in pH when additional acid or base is added to the solution.
  • Salts are ionic compounds that dissociate into cations and anions when dissolved in solution. Salts increase the ionic strength of a solution, and consequently decrease interactions between nucleic acids with other cations.
  • a reverse micelle is destroyed when the micelle no longer exists and the monophase no longer exists.
  • a reverse micelle is destroyed when the micelle is disrupted.
  • the reverse micelle is destroyed by chemically modifying the surfactant so that water in oil emulsion is destroyed and the phases separate.
  • a destructible reverse micelle is a reverse micelle that can be destroyed such that the water in oil emulsion is destroyed and the phases separate.
  • a destructible reverse micelle can undergo a biological, chemical, or biochemical reaction such that the reverse micelle is destroyed. Biological, chemical, or biochemical reactions involve the formation or cleavage of ionic and/or covalent bonds.
  • the destructible reverse micelle contains a surfactant that is cleavable, destroyable, or chemically modifiable.
  • the surfactant can be a disulfide of the general formula A-S-S- B, in which chemical group A is a hydrophobic group and chemical group B is a hydrophilic group.
  • the present invention also relates to a method in which a biologically active compound is delivered to a cell comprising a step in which the biologically active compound is mixed with a biologically-labile surfactant.
  • a biologically-labile surfactant is a surfactant in which the hydrophobic moiety is cleaved from the hydrophilic moiety by cellular processes or its surfactant properties are rendered inactive within the cell, tissue or organism. Examples include surfactants that contain disulfide bonds that are labile within the cell, tissue, or organism.
  • a transfection reagent is a compound or compounds used in the prior art that bind(s) to or complex(es) with polynucleotides and mediates their entry into cells.
  • the transfection reagent also mediates the binding and internalization of polynucleotides into cells.
  • Examples of transfection reagents include cationic liposomes and lipids, calcium phosphate precipitates, and polylysine complexes. At times, the transfection reagent has a net positive charge that binds to the polynucleotide's negative charge.
  • the transfection reagent mediates binding of polynucleotides to cell via its positive charge (that binds to the cell membrane's negative charge) or via ligands that bind to receptors in the cell.
  • cationic liposomes or polylysine complexes have net positive charges that enable them to bind to DNA.
  • Other vehicles are also used, in the prior art, to transfer genes into cells. These include complexing the polynucleotides on particles that are then accelerated into the cell. This is termed biolistic or gun techniques. Other methods include eletroporation in which a device is used to give an electric charge to cells. The charge increases the permeability of the cell.
  • nucleic acid is a term of art that refers to a polymer containing at least two nucleotides.
  • Nucleotides contain a sugar deoxyribose (in DNA) or ribose (in RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate groups.
  • Bases include purines and pyrimidines, which further include natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and synthetic derivatives of purines and pyrimidines, or natural analogs. Nucleotides are the monomeric units of nucleic acid polymers.
  • a "polynucleotide” is distinguished here from an “oligonucleotide” by containing more than 80 monomeric units; oligonucleotides contain from 2 to 80 nucleotides.
  • the term nucleic acid includes deoxyribonucleic acid (“DNA”) and ribonucleic acid (“RNA”).
  • DNA may be in the form of anti-sense, plasmid DNA, parts of a plasmid DNA, vectors (PI, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives of these groups.
  • RNA may be in the form of oligonucleotide RNA, tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), anti-sense RNA, ribozymes, chimeric sequences, or derivatives of these groups.
  • Anti-sense is a nucleic acid that interferes with the function of DNA and/or RNA. This may result in suppression of expression. Natural nucleic acids have a phosphate backbone, artificial nucleic acids may contain other types of backbones, nucleotides, or bases.
  • DNA and RNA may be single, double, triple, or quadruple stranded.
  • “Expression cassette” refers to a natural or recombinantly produced nucleic acid which is capable of expressing protein(s).
  • a DNA expression cassette typically includes a promoter (allowing transcription initiation), and a sequence encoding one or more proteins.
  • the expression cassette may include trancriptional enhancers, non- coding sequences, splicing signals, transcription termination signals, and polyadenylation signals.
  • RNA expression cassette typically includes a translation initiation codon (allowing translation initiation), and a sequence encoding one or more proteins.
  • the expression cassette may include translation termination signals, a polyadenosine sequence, internal ribosome entry sites (IRES), and non- coding sequences.
  • Delivery of a nucleic acid means to transfer a nucleic acid from a container outside a mammal to near or within the outer cell membrane of a cell in the mammal.
  • the term “transfection” may be used, in general, as a substitute for the term “delivery,” or, more specifically, the transfer of a nucleic acid from directly outside a cell membrane to within the cell membrane.
  • the transferred (or "transf ected") nucleic acid may contain an expression cassette. If the nucleic acid is a primary RNA transcript that is processed into messenger RNA, a ribosome translates the messenger RNA to produce a protein within the cytoplasm.
  • nucleic acid If the nucleic acid is a DNA, it enters the nucleus where it is transcribed into a messenger RNA that is transported into the cytoplasm where it is translated into a protein. Therefore if a nucleic acid expresses its cognate protein, then it must have entered a cell. A protein may subsequently be degraded into peptides, which may be presented to the immune system.
  • Polypeptide refers to a linear series of amino acid residues connected to one another by peptide bonds between the alpha-amino group and carboxyl group of contiguous amino acid residues.
  • Protein refers herein to a linear series of greater than 2 amino acid residues connected one to another as in a polypeptide.
  • a "therapeutic" effect of the protein in attenuating or preventing the disease state can be accomplished by the protein either staying within the cell, remaining attached to the cell in the membrane, or being secreted and dissociated from the cell where it can enter the general circulation and blood
  • Secreted proteins that can be therapeutic include hormones, cytokines, growth factors, clotting factors, anti-protease proteins (e.g., alphal-antitrypsin), angiogenic proteins (e.g., vascular endothelial growth factor, fibroblast growth factors), anti- angjogenic proteins (e.g., endostatin, angiostatin), and other proteins that are present in the blood.
  • Proteins on the membrane can have a therapeutic effect by providing a receptor for the cell to take up a protein or lipoprotein (e.g., low density lipoprotein receptor).
  • Therapeutic proteins that stay within the cell can be enzymes that clear a circulating toxic metabolite as in phenylketonuria. They can also cause a cancer cell to be less proliferative or cancerous (e.g., less metastatic), or interfere with the replication of a virus.
  • Intracellular proteins can be part of the cytoskeleton (e.g., actin, dystrophin, myosins, sarcoglycans, dystroglycans) and thus have a therapeutic effect in cardiomyopathies and musculoskeletal diseases (e.g., Duchenne muscular dystrophy, limb-girdle disease).
  • Other therapeutic proteins of particular interest to treating heart disease include polypeptides affecting cardiac contractility (e.g., calcium and sodium channels), inhibitors of restenosis (e.g., nitric oxide synthetase), angiogenic factors, and anti-angiogenic factors.
  • Biomolecule refers to peptides, polypeptides, proteins, enzymes, polynucleotides, oligonucleotides, viruses, antigens, carbohydrates (and congugates), lipids, and sacharides.
  • Enzymes are proteins evolved by the cells of living organisms for the specific function of catalyzing chemical reactions.
  • a chemical reaction can take place within the micelle and nucleic acid complex.
  • a chemical reaction is defined as the formation or cleavage of covalent or ionic bonds.
  • a polymer can be formed.
  • a polymer is defined as a compound containing more than two monomers.
  • a monomer is a compound that can be attached to itself or another monomer and thus form a polymer.
  • the surfactant is polymerized by chain or step polymerization and then the surfactant properties are destroyed. This destruction of the surfactant properties could be a accomplished by breaking a chemical bond and separating the hydrophilic and hydrophobic moities.
  • a chemical reaction can be used to attach a gene transfer enhancing signal to the nucleic acid complex.
  • the gene transfer enhancing signal (or abbreviated as the Signal) is defined in this specification as a molecule that modifies the nucleic acid complex and can direct it or the nucleic acid to a cell location (such as tissue) or location in a cell (such as the nucleus) either in culture or in a whole organism. By modifying the cellular or tissue location of the foreign gene, the expression of the foreign gene can be enhanced.
  • the gene transfer enhancing signal can be a protein, peptide, lipid, steroid, sugar, carbohydrate, (non-expresssing) nucleic acid or synthetic compound.
  • the gene transfer enhancing signals enhance cellular binding to receptors, cytoplasmic transport to the nucleus and nuclear entry or release from endosomes or other intracellular vesicles.
  • Nuclear localizing signals enhance the targeting of the gene into proximity of the nucleus and/or its entry into the nucleus.
  • Such nuclear transport signals can be a protein or a peptide such as the SV40 large T ag NLS or the nucleoplasmin NLS.
  • These nuclear localizing signals interact with a variety of nuclear transport factors such as the NLS receptor (karyopherin alpha) which then interacts with karyopherin beta
  • the nuclear transport proteins themselves could also function as NLS's since they are targeted to the nuclear pore and nucleus.
  • Signals that enhance release from intracellular compartments can cause DNA release from intracellular compartments such as endosomes (early and late), lysosomes, phagosomes, vesicle, endoplasmic reticulum, golgi apparatus, trans golgi network (TGN), and sarcoplasmic reticulum. Release includes movement out of an intracellular compartment into cytoplasm or into an organelle such as the nucleus. Releasing signals include chemicals such as chloroquine, bafilomycin or Brefeldin Al and the ER-retaining signal (KDEL sequence), viral components such as influenza virus hemagglutinin subunit HA-2 peptides and other types of amphipathic peptides.
  • Cellular receptor signals are any signal that enhances the association of the gene with a cell. This can be accomplished by either increasing the binding of the gene to the cell surface and/or its association with an intracellular compartment, for example: ligands that enhance endocytosis by enhancing binding the cell surface. This includes agents that target to the asialoglycoprotein receptor by using asiologlycoproteins or galactose residues. Other proteins such as insulin, EGF, or transferrin can be used for targeting. Peptides that include the RGD sequence can be used to target many cells. Chemical groups that react with sulfhydryl or disulfide groups on cells can also be used to target many types of cells. Folate and other vitamins can also be used for targeting. Other targeting groups include molecules that interact with membranes such as lipids fatty acids, cholesterol, dansyl compounds, and amphotericin derivatives. In addition viral proteins could be used to bind cells.
  • a polynucleotide can be delivered to a cell in order to produce a cellular change that is therapeutic.
  • the delivery of polynucleotides or other genetic material for therapeutic purposes is gene therapy.
  • the polynucleotides are coded to express a whole or partial protein, or may be anti-sense, and can be delivered either directly to the organism in situ or indirectly by transfer to a cell that is then transplanted into the organism.
  • the protein can be missing or defective in an organism as a result of genetic, inherited or acquired defect in its genome.
  • a polynucleotide may be coded to express the protein dystrophin that is missing or defective in Duchenne muscular dystrophy.
  • the coded polynucleotide is delivered to a selected group or groups of cells and incorporated into those cell's genome or remain apart from the cell's genome. Subsequently, dystrophin is produced by the formerly deficient cells.
  • Other examples of imperfect protein production that can be treated with gene therapy include the addition of the protein clotting factors that are missing in the hemophilia's and enzymes that are defective in inborn errors of metabolism such as phenylalanine hydroxylase.
  • a delivered polynucleotide can also be therapeutic in acquired disorders such as neurodegenerative disorders, cancer, heart disease, and infections.
  • the polynucleotide has its therapeutic effect by entering the cell. Entry into the cell is required for the polynucleotide to produce the therapeutic protein, to block the production of a protein, or to decrease the amount of a RNA.
  • a therapeutic effect of the protein in attenuating or preventing the disease state can be accomplished by the protein either staying within the cell, remaining attached to the cell in the membrane or being secreted and dissociating from the cell where it can enter the general circulation and blood.
  • Secreted proteins that can be therapeutic include hormones, cytokines, growth factors, clotting factors, anti-protease proteins (e.g. alpha-antitrypsin) and other proteins that are present in the blood. Proteins on the membrane can have a therapeutic effect by providing a receptor for the cell to take up a protein or lipoprotein.
  • the low density lipoprotein (LDL) receptor could be expressed in hepatocytes and lower blood cholesterol levels and thereby prevent atherosclerotic lesions that can cause strokes or myocardial infarction.
  • Therapeutic proteins that stay within the cell can be enzymes that clear a circulating toxic metabolite as in phenylketonuria. They can also cause a cancer cell to be less proliferative or cancerous (e.g. less metastatic). A protein within a cell could also interfere with the replication of a virus.
  • the delivered polynucleotide can stay within the cytoplasm or nucleus apart from the endogenous genetic material.
  • the polynucleotide could recombine (become a part of) the endogenous genetic material.
  • DNA can insert into chromosomal DNA by either homologous or non-homologous recombination.
  • Parenchymal cells are the distinguishing cells of a gland or organ contained in and supported by the connective tissue framework.
  • the parenchymal cells typically perform a function that is unique to the particular organ.
  • the term "parenchymal” often excludes cells that are common to many organs and tissues such as fibroblasts and endothelial cells within the blood vessels.
  • the parenchymal cells include hepatocytes, Kupffer cells and the epithelial cells that line the biliary tract and bile ductules.
  • liver parenchyma The major constituent of the liver parenchyma are polyhedral hepatocytes (also known as hepatic cells) that presents at least one side to an hepatic sinusoid and apposed sides to a bile canaliculus. Liver cells that are not parenchymal cells include cells within the blood vessels such as the endothelial cells or fibroblast cells.
  • the parenchymal cells include myoblasts, satellite cells, myotubules, and myofibers.
  • the parenchymal cells include the myocardium also known as cardiac muscle fibers or cardiac muscle cells and the cells of the impulse connecting system such as those that constitute the sinoatrial node, atrioventricular node, and atrioventricular bundle.
  • the parenchymal cells include cells wilhin the acini such as zymogenic cells, centroacinar cells, and basal or basket cells and cells within the islets of Langerhans such as alpha and beta cells.
  • the parenchymal cells include reticular cells and blood cells (or precursors to blood cells) such as lymphocytes, monocytes, plasma cells and macrophages.
  • the parenchymal cells include neurons, glial cells, microglial cells, oligodendrocytes, Schwann cells, and epithelial cells of the choroid plexus.
  • parenchymal cells include cells of collecting tubules and the proximal and distal tubular cells.
  • the parenchyma includes epithelial cells.
  • the parenchymal cells include cells that produce hormones.
  • the parenchymal cells include the principal cells (chief cells) and oxyphilic cells.
  • the parenchymal cells include follicular epithelial cells and parafollicular cells.
  • the parenchymal cells include the epithelial cells within the adrenal cortex and the polyhedral cells within the adrenal medulla.
  • the parenchymal cells include epithelial cells, glandular cells, basal, and goblet cells.
  • the parenchymal cells include the epithelial cells, mucus cells, goblet cells, and alveolar cells.
  • the parenchymal cells include adipose cells or adipocytes.
  • the parenchymal cells include the epithelial cells of the epidermis, melanocytes, cells of the sweat glands, and cells of the hair root.
  • the parenchyma includes chondrocytes.
  • the parenchyma includes osteoblasts, osteocytes, and osteoclasts.
  • Intravascular refers to an intravascular route of administration that enables a polymer, oligonucleotide, or polynucleotide to be delivered to cells more evenly distributed and more efficiently than direct injections.
  • Intravascular herein means within an internal tubular structure called a vessel that is connected to a tissue or organ within the body of an animal, including mammals.
  • a bodily fluid flows to or from the body part.
  • bodily fluid include blood, lymphatic fluid, or bile.
  • vessels include arteries, arterioles, capillaries, venules, sinusoids, veins, lymphatics, and bile ducts.
  • the intravascular route includes delivery through the blood vessels such as an artery or a vein.
  • “Intracoronary" refers to an intravascular route for delivery to the heart wherein the blood vessels are the coronary arteries and veins.
  • Permeability is defined herein as the propensity for macromolecules such as nucleic acids to move through vessel walls and enter the extravascular space.
  • One measure of permeability is the rate at which macromolecules move through the vessel wall and out of the vessel.
  • Another measure of permeability is the lack of force that resists the movement through the vessel wall and out of the vessel.
  • Vessels contain elements that prevent macromolecules from leaving the intravascular space (internal cavity of the vessel). These elements include endothelial cells and connective material (e.g., collagen). High permeability indicates that there are fewer of these elements that can block the egress of macromolecules and that the spaces between these elements are larger and more numerous. In this context, high permeability enables a high percentage of nucleic acids being delivered to leave the intravascular space, while low permeability indicates that a low percentage of the nucleic acids will leave the intravascular space.
  • the permeability of a blood vessel can be increased by increasing the intravascular hydrostatic pressure.
  • the intravascular hydrostatic pressure is increased by rapidly (from 1 seconds to 30 minutes) injecting a nucleic acid in solution into the blood vessel, which increases the hydrostatic pressure.
  • hydrostatic pressure is increased by obstructing the outflow of the injection solution from the tissue for a period of time sufficient to allow delivery of a nucleic acid. Obstructing means to block or impede the outflow of injection fluid, thereby transiently (reversibly) blocking the outflow of the blood.
  • rapid injection may be combined with obstructing the outflow in yet another preferred embodiment.
  • an afferent vessel supplying an organ is rapidly injected while the efferent vessel draining the tissue is blocked transiently (e.g., by ligation, or by an inflated intravascular balloon).
  • the efferent vessel also called the venous outflow or tract
  • draining outflow from the tissue is partially or totally clamped for a period of time sufficient to allow delivery of a nucleic acid.
  • an efferent vessel is injected while the corresponding afferent vessel is occluded.
  • An administration route involving the mucosal membranes is meant to include nasal, bronchial, inhalation into the lungs, or via the eyes.
  • Transdermal refers to application to mammal skin in which drug delivery occurs by crossing the dermal layer.
  • Crosslinking refers to the chemical attachment of two or more molecules with a bifunctional reagent.
  • a bifunctional reagent is a molecule with two reactive ends. The reactive ends can be identical as in a homobifunctional molecule, or different as in a heterobifucnctional molecule.
  • Electrostatic interactions are the non-covalent association of two or more substances due to attractive forces between positive and negative charges.
  • Amphipathic compounds have both hydrophilic (water-soluble) and hydrophobic (water-insoluble) parts.
  • a polycation is a polymer containing a net positive charge, for example poly-L-lysine hydrobromide.
  • the polycation can contain monomer units that are charge positive, charge neutral, or charge negative, however, the net charge of the polymer must be positive.
  • a polycation also can mean a non-polomeric molecule which contains two or more positive charges.
  • a polyanion is a polymer containing a net negative charge, for example polyglutamic acid. The polyanion can contain monomer units that are charge negative, charge neutral, or charge positive, however, the net charge on the polymer must be negative.
  • a polyanion can also mean a non-polomeric molecule that contains two or more negative charges.
  • poly ion includes polycation, polyanion, zwiterionic polymers, and neutral polymers.
  • zwiterionic refers to the product (salt) of the reaction between an acidic group and a basic group that are part of the same molecule.
  • Mixing means the method of interdispursing two or more solvents, or solvent(s) and solute(s). Sonication and agitation are forms of mixing.
  • Solvent refers to a material in the liquid phase that can be used to solubilize (dissolve) a compound.
  • Solute refers to a compound dissolved in a solvent.
  • Hydrocarbon means containing carbon and hydrogen atoms; and halohydrocarbon means containing carbon, halogen (F, Cl, Br, I), and hydrogen atoms.
  • Alkyl means containing sp3 hybridized carbon atoms; alkenyl means containing two or more sp2 hybridized carbon atoms; aklkynyl means containing two or more sp hybridized carbon atoms; aralkyl means containing one or more aromatic ring(s) in addition containing sp3 hybridized carbon atoms; aralkenyl means containing one or more aromatic ring(s) in addition to containing two or more sp2 hybridized carbon atoms; aralkynyl means containing one or more aromatic ring(s) in addition to containing two or more sp hybridized carbon atoms; steroid includes natural and unnatural steroids and steroid derivatives.
  • carbohydrates include natural and unatural sugars (for
  • Hydrophilic groups indicate in qualitative terms that the chemical moiety is water- preferring. Typically, such chemical groups are water soluble, and are hydrogen bond donors or acceptors with water. Examples of hydrophilic groups include compounds with the following chemical moieties carbohydrates; polyoxyethylene, peptides, oligonucleotides and groups containing amines, amides, alkoxy amides, carboxylic acids, sulfurs, or hydroxyls. Hydrophobic groups indicate in qualitative terms that the chemical moiety is water-avoiding. Typically, such chemical groups are not water soluble, and tend not to hydrogen bond. Hydrocarbons are hydrophobic groups.
  • terapéutica and “therapeutic results” are defined in this application as levels of gene products, including reporter (marker) gene products, which indicate a reasonable expectation of gene expression using similar compounds (other nucleic acids including other genes), at levels considered sufficient by a person having ordinary skill in the art of gene therapy.
  • Hemophilia A and B are caused by deficiencies of the X-linked clotting factors VHI and DC, respectively. Their clinical course is greatly influenced by the percentage of normal serum levels of factor VH1 or DC: ⁇ 2%, severe; 2-5%, moderate; and 5-30% mild. This indicates that in severe patients only 2% of the normal level can be considered therapeutic. Levels greater than 6% prevent spontaneous bleeds but not those secondary to surgery or injury.
  • a person having ordinary skill in the art of gene therapy would reasonably anticipate therapeutic levels of expression of a gene specific for a disease based upon sufficient levels of marker gene results.
  • marker genes were expressed to yield a protein at a level comparable in volume to 2% of the normal level of factor Vrfl, it can be reasonably expected that the gene coding for factor " VTTI would also be expressed at similar levels.
  • reporter gene/protein systems There are three types of reporter (marker) gene products that are expressed from reporter genes.
  • the reporter gene/protein systems include:
  • intracellular gene products such as luciferase, ⁇ -galactosidase, or chloramphenicol acetyl transferase. Typically, they are enzymes whose enzymatic activity can be easily measured.
  • Intracellular gene products such as ⁇ -galactosidase or green fluorescent protein which identify cells expressing the reporter gene. On the basis of the intensity of cellular staining, these reporter gene products also yield qualitative information concerning the amount of foreign protein produced per cell.
  • Secreted gene products such as growth hormone, factor DC, or alphal-antitrypsin are useful for determining the amount of a secreted protein that a gene transfer procedure can produce.
  • the reporter gene product can be assayed in a small amount of blood.
  • Example 2 Demonstration of Micelle Formation with ⁇ -D-Glucopyranosyl dodecane disulfide, and Micelle Destruction with Dithiothreitol: To a solution of ⁇ -D-Qucopyranosyl dodecane disulfide (10 mg) in 1 mL CD 3 C1 was added 1 mL H 2 O The sample was rapidly mixed resulting in a thick white emulsion. After 18 h, the organic and aqueous layers were emulsified to approximately 95%. After 4 d, the organic and aqueous layers remained emulsified to approximately 70%.
  • PCILuc DNA (pDNA) (Zhang, G., Vargo, D., Budker, V., Armstrong, N., Knechtie, S., Wolff, J. A. Human Gene Therapy, 8, 1763-1772, 1997) (11 ⁇ g) was taken up in a solution (3-67 ⁇ L) of HEPES (25 mM, pH 7.8) and EDTA (0.5 mM).
  • Polyoxyethylene(4) lauryl ether (Brij 30) (1.2 mL, Aldrich Chemical Company) was taken up in 2,2,4-trimethylpentane (TMP) (8.8 mL, Aldrich Chemical Company). To the Brij 30/TMP solution (0.7 mL) was added the pDNA in buffer (3-67 ⁇ L).
  • HLB hydrophile-lipophile balance
  • PCILuc DNA (pDNA) (Zhang, G., Vargo, D., Budker, V., Armstrong, N., Knechtie, S., Wolff, J. A. Human Gene Therapy, 8, 1763-1772, 1997) (36 ⁇ g) was taken up in a solution of HEPES (25 m pH 7.8) and EDTA (0.5 mM) (10 ⁇ L, 20 ⁇ L, 30 ⁇ L, and 50 ⁇ L). The resulting solutions were added to a mixture of Polyoxyethylene(4) lauryl ether (Brij 30) (Aldrich Chemical Company)/ 2,2,4-trimethylpentane (TMP) (Aldrich Chemical Company) (1 mL, 1:7.3 v/v) and agitated.
  • Brij 30 Polyoxyethylene(4) lauryl ether
  • TMP 2,2,4-trimethylpentane
  • the UV adsorption was determined (Perkin Elmer, UV/VIS Spectrophotometer, model Lambda 6) against 10 ⁇ L of HEPES (25 mM, pH 7.8) and EDTA (0.5 mM) buffer in Brij 30 TMP (1 mL, 1:7.3 v/v). The samples were centriiuged 5 min at 15000 ⁇ m and the UV adso ⁇ tion was again determined.
  • micelles Two types appear to be present in the samples. There are small, "empty” micelles, and large pDNA containing micelles. It appears that the size of micelles containing pDNA increases as the concentration of pDNA increases. The micelle appears to be saturated at a size of 50-60 nm.
  • pDNA was modified to a level of 1 Rhodamine per 100 bases using Mirus' Labellt ® Rhodamine kit (Rhodamine Containing DNA Labeling Reagent, Mirus Co ⁇ oration).
  • the modified pDNA (2.5 ⁇ g) was solubilized in different volumes of HEPES (25 niM, pH 7.8) and EDTA (0.5 mM ) and added to a solution of Brij 30/TMP (0.7 mL, 1:7.3 v/v), and agitated.
  • the fluorescence was determined using a fluorescence spectrophotometer (Hitachi, Model F-3010), at an excitation wavelength of 591 nm, and an emission wavelength of 610 nm.
  • pDNA was modified to a level of 1 Rhodamine per 100 bases using standard procedures (Labellt ® , Mirus Co ⁇ oration). Labeled pDNA (various amounts) was taken up in HEPES (25 mM, pH 7.8) EDTA (0.5 mM ) (various amounts) and was mixed with unmodified pDNA (various amounts) to afford 2.5 ⁇ g total of pDNA. The resulting solution was added to Brij 30/TMP (0.7 mL, 1:7.3 v/v) and the fluorescence was determined using a fluorescence spectrophotometer (Hitachi, Model F-3010), at an excitation wavelength of 591 nm, and an emission wavelength of 610 nm..
  • the fluorescence was also determined for the similar ratios of Rh-labeled pDNA/pDNA containing 2 mM spermidine (Sigma Chemical Company) in HEPES (25 m pH 7.8) and EDTA (0.5 mM) (0.7 mL).
  • the fluorescense data indicates a relatively weak affect of Rh-labeled pDNA dilution by unlabeled pDNA.
  • a strong effect of the Rh-pDNA dilution by unlabeled DNA is shown.
  • the pDNA condensation starts from monomolecular condensation and therefore show little effect by the dilution protocol.
  • the strong effect indicates that condensation is multimolecular.
  • a sample containing pDNA in HEPES (25 mM, pH 7.8) and EDTA (0.5 mM) (20 ⁇ L) and TMP (1 mL) demonstrated ring like structures with an external diameter of 59.8 ⁇ 12.5 nm and an internal diameter of 32.9 ⁇ 12.1 nm.
  • the volume of the terroid ring V ( ⁇ 24 )(Rout-Rin) 2 (Rout + Rin) equal 41*10 3 nm 3 .
  • the volume of "dry" PCILuc DNA is 6.4*10 3 nm 3 . With consideration of packing parameter every toroid therefore contains five pDNA's.
  • NBD-PLL (example 9) was mixed with Polyoxyethylene(4) lauryl ether (Brij 30) (Aldrich Chemical Company)/ 2,2,4-trimethylpentane (TMP) (Aldrich Chemical Company) (1 :7.3 v/v), and then mixed with an equal volume of Brij 30/TMP (1 :7.3 v/v) that contained either HEPES (25 nrM, pH 7.8) and EDTA (0.5 mM) or HEPES (25 mM pH 7.8) and EDTA (0.5 mM) with pDNA (various amounts) After 10 min at ambient temperature, the fluorescence was determined for each sample.
  • the solution was heated to 40 °C for 25 min at which time HEPES (25 mM, pH 7.8), EDTA (0.5 mM), and NaCl (100 mM) (200 ⁇ L) was added, followed by EtOH (50 ⁇ L) and EtOAc (0.5 mL). After mixing and centrifugation, the aqueous layer was washed with EtOAc (2 x 1 mL) and Ether (2 x 1 mL). The samples were spun (5 min, 12000 ⁇ m) and dialyzed for 16 h against HEPES (25 mM, pH 7.8) and NaCl (100 mM). The UV abso ⁇ tion was determined (Perkin Elmer UV7VIS Spectrophotometer, Model Lambda 6).
  • the pDNA in the extracted complexes is compacted because it does not interact with the fluorescent intercolator TO6.
  • Example 12 PCILuc DNA Polyethylenimine Complexes in Reverse Micelles.
  • pDNA was labeled as above (Labellt ® , Minis Co ⁇ oration). Labeled pDNA (14 ⁇ g) was taken up in HEPES (25 rriM, pH 7.8) and EDTA (0.5 mM) (various amounts) and added to Polyoxyethylene(4) lauryl ether (Brij 30) (Aldrich Chemical Company )/2,2,4- trimethylpentane (TMP) (Aldrich Chemical Company) (1 mL, 1:7.3 v/v). The fluorescence and turbidity of each sample was determined.
  • PEI Polyethylenimine

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Abstract

L'invention concerne un complexe assurant l'administration à des cellules et consistant, plus particulièrement, à administrer un acide nucléique dans un micelle inversée. Cette micelle a la propriété de rendre l'acide nucléique plus compacte afin de faciliter son administration. D'autres molécules permettent d'exercer une interaction avec le complexe acide nucléique-micelle pour améliorer l'administration comme par exemple une liaison disulfure.
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