WO1999055825A1 - Technique permettant de fabriquer un compose en formant un polymere a partir d'un medicament matrice - Google Patents

Technique permettant de fabriquer un compose en formant un polymere a partir d'un medicament matrice Download PDF

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WO1999055825A1
WO1999055825A1 PCT/US1999/008965 US9908965W WO9955825A1 WO 1999055825 A1 WO1999055825 A1 WO 1999055825A1 US 9908965 W US9908965 W US 9908965W WO 9955825 A1 WO9955825 A1 WO 9955825A1
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dna
polymer
polymerization
template
complexes
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PCT/US1999/008965
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Jon A. Wolff
James E. Hagstrom
Vladimir G. Budker
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Mirus Corporation
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Priority to EP99920014A priority Critical patent/EP1073707A4/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • 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

  • 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-poly cations complexes It was shown that catiomc proteins like histones and protamines or synthetic polymers like polylysine, polyargmme, polyornithine, DEAE dextran, polybrene, and polyethylenimine were effective intracellular delivery agents while small polycations like spermine were ineffective (Feigner, P L (1990) Advanced Drug Delivery Rev 5, 163-187, Boussif, O , Lezoualch, F , Zanta, M A , Mergny, M D , Scherman, D , Demeneix, B , & Behr, J P (1995) Proc Natl Ac
  • Polycations provide attachment of DNA to the target cell surface: The polymer forms a cross-b ⁇ dge between the polyanionic nucleic acids and the polyanionic surfaces of the cells As a result the mam mechanism of DNA translocation to the intracellular space might be non-specific adsorptive endocytosis which may be more effective then liquid endocytosis or receptor-mediated endocytosis Furthermore, 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 ( Perales, J C , Ferkol, T Molas, M & Hanson, W (1994) Eur J Biochem 226, 255-266, Cotten, M, WagnerJE & Birnstiel, M L (1993) Methods in Enzymology 217, 618- 644, Wagner, E , Cunel, D , & Cotten, M (1994) Advanced Drug Delivery Rev 1 14, 113-135) 2) Polycations protect DNA in complexes against nucleas
  • DNA/polymer complex is cntical for gene delivery m vivo
  • DNA needs to cross the endothelial bar ⁇ er and reach the parenchymal cells of interest
  • the largest endotheha fenestrae (holes in the endothelial barner) occurs m the liver and have an average diameter 100 nm
  • the fenestrae size in other organs is much lower
  • the size of the DNA complexes is also important for the cellular uptake process After binding to the target cells the DNA- polycation complex should be taken up by endocytosis Since the endocytic vesicles have a homogenous internal diameter of about 100 nm in hepatocytes of similar size in other cell types, the DNA complexes need to be smaller than 100 nm (Geuzze, H.J , Slot, J.
  • Toroids have been considered an attractive form for gene delivery because they have the lowest size. While the size of DNA toroids produced within single preparations has been shown to vary considerably, toroid size is unaffected by the length of DNA being condensed. DNA molecules from 400 bp to genomic length produce toroids similar in size (Bloomfield, V.A. (1991) Biopolymers 31, 1471-1481). Therefore one toroid can include from one to several DNA molecules. The kinetics of
  • DNA collapse by polycations which resulted in toroids is very slow.
  • DNA condensation by Co(NH3)6CI3 needs 2 hours at room temperature (Arscott, P G , Ma, C , & Bloomfield, V A (1995) Biopolymers 36, 345-364)
  • a process for drug delivery is described in which polymerization and chemical reaction processes are induced in the presence of the drug in order to deliver the drug or biologically active compound.
  • Drug delivery encompasses the delivery of a biologically active compound to a cell.
  • delivering we mean that the drug becomes associated with the cell.
  • the drug can be on the membrane of the cell or inside the cytoplasm, nucleus, or other organelle of the cell.
  • transfection or the process of "transfecting” and also it has been termed “transformation”.
  • a biologically active compound is a compound having the potential to react with biological components. Pharmaceuticals, proteins, peptides and nucleic acids are examples of biologically active compounds.
  • the template polymer can be a polyanion such as a nucleic acid.
  • 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”.
  • a new method is described for forming condensed nucleic acid by having a chemical reaction take place in the presence of the nucleic acid.
  • a process is also described of forming in the presence of the nucleic acid a polymer that has affinity to nucleic acid.
  • a process is described of forming an interpolyelectrolyte complex containing nucleic acids by having a chemical reaction take place in the presence of the nucleic acid.
  • the nucleic acid-binding polymer can form as a result of template polymerization. This obviously excludes the formation of polymers such as proteins or nucleic acids or other derivatives that bind nucleic acid by Watson-Crick binding.
  • nucleic acid Previously, the occurrence of chemical reactions or the process of polymerization in the presence of the nucleic acid has been assiduously avoided when delivering nucleic acid. Perhaps, this arose out of concerns that the processes of chemical reactions or polymerization would chemically modify the nucleic acid and thereby render it not biologically active. Surprisingly, we show that we can perform polymerizations in the presence of nucleic acids without chemically modifying the nucleic acid and that the nucleic acid is still functional. For example, a plasmid construct containing a promoter and the reporter gene luciferase can still express as much luciferase as native plasmid after transfection into cells.
  • the process of forming a polymer in the presence of nucleic acid has several advantages.
  • Figure 1 illustrates, aggregation and precipitation of the nucleic acid can be avoided by having the polymerization take place in the presence of the nucleic acid.
  • This newly described process enabled us to form supramolecular complexes of nucleic acid and polymer rapidly, consistently, and at very high concentrations of polynucleic acid. In fact, high concentration of the template nucleic acid favors this process.
  • the previously described process of mixing a nucleic acid and an already-formed polycation such as polylysine
  • the previously-described procedure requires that the mixing, salt and ionicity conditions must be carefully controlled as well. This explains why the use of polylysine-DNA complexes are not widely used for the transfer of DNA into cells and is only done in a few laboratories.
  • the other advantage that flows from the newly described process of having polymerization take place in the presence of nucleic acid is that polymers could form that would not be able to become associated with nucleic acids if the polymer was formed first.
  • the polymerization process could result in a hydrophobic polymer that is not soluble in aqueous solutions unless it is associated with nucleic acid.
  • a hydrophobic moiety comprises a C6-C24 alkane, C6-C24 alkene, sterol, steroid, lipid, or hydrophobic hormone.
  • the process of having the polymerization taking place in organic solvents and heterophase systems enables more types and more defined types of vesicles to be formed.
  • nucleic acid/polymer complexes will be smaller.
  • the size of DNA/polymer complex is critical for gene delivery especially in vivo.
  • nucleic acids can be used for transferring nucleic acids into cells or an organism such as for drug delivery. They may also be used for analytical methods or the construction of new materials. They may also be used for preparative methods such as in the purification of nucleic acids. They are also useful for many types of recombinant DNA technology. For example, they may be used to generate sequence binding molecules and protect specific sequences from nuclease digestion. Protection of specific regions of DNA is useful in many applications for recombinant DNA technology.
  • a preferred embodiment provides a method of making a compound for delivery to a cell, comprising: forming a polymer in the presence of a biologically active drug.
  • Another preferred embodiment provides a method of making a compound for delivery to a cell, comprising: cross-linking a polymer in the presence of a polyion, thereby forming a complex of polymer and polyion; and, delivering the complex to the 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 polyion thereby providing a deliverable polyion.
  • Another preferred embodiment provides a method of making a compound for delivery to a cell, comprising: mixing a polyion with a first polymer and a second polymer thereby forming a deliverable complex.
  • FIG. 1 is a comparison of pDNA following template polymerization and complexation contrasted with preformed polycations binding and precipitating.
  • FIG. 2 shows complexes ranging in size from 40 - 70 nanometers in diameter after being dried onto carbon grids and stained with methylamine tungstate.
  • FIG. 3 illustrates template dependent polymerization of NLS Peptides using SDS- PAGE.
  • FIG. 4 illustrates the relationship between turbidity (an indication of aggregation) and the molar charge ratio of polylysine (PLL): plasmid DNA without and with 100 mM NaCI and without and without template polymerization/caging (indicated by +DTBP).
  • FIG. 5 illustrates the ability of dextran sulfate (DS) to enable ethidium bromide to interact with plasmid DNA that has been complexed with varying ratios of PLL/DNA (molar ratio of lysine residue to DNA base) (numbers in the legends) with or without the addition of DTBP.
  • DS dextran sulfate
  • FIG. 6 illustrates the effect of varying the histone/DNA ratio on the sizes of histone/DNA complexes with the addition of DTBP.
  • a process for drug delivery is described in which polymerization and chemical reaction processes take place in the presence of the drug in order to deliver the drug.
  • the polymer is formed from a variety of monomers in the presence of the drug and then the mixture is delivered. The mixture could undergo further purification or preparative methods.
  • Drug delivery encompasses the delivery of a biologically active compound to a cell. This can be accomplished with prokaryotic or eukaryotic cell. It includes mammalian cells that are either outside or within an organism.
  • the drug also includes the administration of the drug to the whole organism by standard routes such as intravenous, intra-arterial, intra-bile duct, intramuscular, subcutaneous, intraperitoneal, or direct injections into tissues such as the liver, brain, kidneys, heart, eyes, lymph nodes, bone, gastrointestinal tract. It also includes delivery into vessels such as blood, lymphatic, biliary, renal, or brain ventricles.
  • standard routes such as intravenous, intra-arterial, intra-bile duct, intramuscular, subcutaneous, intraperitoneal, or direct injections into tissues such as the liver, brain, kidneys, heart, eyes, lymph nodes, bone, gastrointestinal tract. It also includes delivery into vessels such as blood, lymphatic, biliary, renal, or brain ventricles.
  • this process is used to deliver nucleic acids.
  • the process of delivering nucleic acids means exposing the cell to the polynucleic in the presence of the delivery system.
  • Cells indicate both prokaryotes and eukaryotes.
  • the cell is located in a living organism and exposing is accomplished by administering the nucleic acid and the delivery system to the organism. It also means mixing the nucleic acids with cells in culture or administering the nucleic acids to a whole organism.
  • Delivering nucleic acids encompasses transfecting a cell with a nucleic acid.
  • These delivery processes include standard injection methods such as intramuscular, subcutaneous, intraperitoneal, intravenous, and intra-arterial. It also includes injections into any vessel such as the bile duct and injections into any tissue such as liver, kidney, brain, thymus, heart, eye, or skin.
  • Drugs, pharmaceuticals, proteins, peptides and nucleic acids are biologically active compounds.
  • the drug can be either the template polymer or the daughter polymer.
  • the template polymer is a polyion, a macromolecule carrying a string of charges, such as a nucleic acid which would be termed a polyanion because of its average negative charge.
  • nucleic acid is a term of art that refers to a string of at least two base-sugar-phosphate combinations. Nucleotides are the monomeric units of nucleic acid polymers.
  • nucleic acid includes both oligonucleic acids and polynucleic acids. Polynucleic acids are distinguished from oligonucleic acid by containing more than 120 monomeric units. In the case of the transfer of nucleic acids into cells, the nucleic acid is the template.
  • the nucleic acid could also 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 delivered polynucleotide could produce a therapeutic protein such as a hormone, cytokine, or growth factor.
  • the polynucleotide in the form of a plasmid DNA could produce the human growth hormone.
  • the polynucleotide could produce an enzyme that is deficient or defective in patients with an inborn error of metabolism.
  • a plasmid DNA could produce phenylalanine hydroxylase which would be therapeutic in patients with phenylketonuria.
  • the polynucleotide could supply an anti-sense that would be therapeutic in patients with a tumor, cancer, or infection.
  • the polynucleotide could be a DNA that is transcribed into an anti-sense molecule.
  • a polymer is a molecule built up by repetitive bonding together of smaller units called monomers.
  • the term polymer includes both oligomers which have two to -80 monomers and polymers having more than 80 monomers.
  • the polymer can be linear, branched network, star, comb, or ladder types of polymer.
  • the polymer can be a homopolymer in which a single monomer is used or can be copolymer in which two or more monomers are used. Types of copolymers include alternating, random, block and graft.
  • a steric stabilizer which is a long chain hydrophilic group that prevents aggregation of final polymer by sterically hindering particle to particle electrostatic interactions.
  • examples include: alkyl groups, PEG chains, polysaccharides, hydrogen molecules, alkyl amines.
  • polymerization processes there are several categories of polymerization processes that can be utilized in the described process.
  • the polymerization can be chain or step. This classification description is more often used that the previous terminology of addition and condensation polymer. "Most step- reaction polymerizations are condensation processes and most chain-reaction polymerizations are addition processes” (M. P. Stevens Polymer Chemistry: An Introduction New York Oxford University Press 1990).
  • step polymerization the polymerization occurs in a stepwise fashion. Polymer growth occurs by reaction between monomers, oligomers and polymers. No initiator is needed since there is the same reaction throughout and there is no termination step so that the end groups are still reactive. The polymerization rate decreases as the functional groups are consumed.
  • step polymerization is done either of two different ways.
  • the monomer has both reactive functional groups (A and B) in the same molecule so that
  • A-A + B-B yields -[A-A-B-B]-
  • these reactions can involve acylation or alkylation.
  • Acylation is defined as the introduction of an acyl group (-COR) onto a molecule.
  • Alkylation is defined as the introduction of an alkyl group onto a molecule.
  • B can be (but not restricted to) an isothiocyanate, isocyanate, acyl azide, N-hydroxysuccinimide, sulfonyl chloride, aldehyde (including formaldehyde and glutaraldehyde), epoxide, carbonate, imidoester, carboxylate, or alkylphosphate, arylhalides (difluoro-dinitrobenzene) or succinic anhyride.
  • function B can be acylating or alkylating agent.
  • function B can be (but not restricted to) an iodoacetyl derivative, maleimide, aziridine derivative, acryloyl derivative, fluorobenzene derivatives, or disulfide derivative (such as a pyridyl disulfide or 5-thio- 2-nitrobenzoic acid ⁇ TNB ⁇ derivatives).
  • function B can be (but not restricted to) a diazoacetate or an amine in which carbonyldiimidazole or carbodiimide is used.
  • function B can be (but not restricted to) an epoxide, oxirane, or an amine in which carbonyldiimidazole or carbodiimide or N, N'- disuccinimidyl carbonate, or N-hydroxysuccinimidyl chloroformate is used.
  • function B can be (but not restricted to) an hydrazine, hydrazide derivative, aldehyde (to form a Schiff Base that may or may not be reduced by reducing agents such as NaCNBH 3 ).
  • A-A plus another agent yields -[A-A]-.
  • function A is a sulfhydryl group then it can be converted to disulfide bonds by oxidizing agents such as iodine (I 2 ) or NaIO4 (sodium periodate), or oxygen (O 2 ).
  • Function A can also be an amine that is converted to a sulfhydryl group by reaction with 2-Iminothiolate (Traut's reagent) which then undergoes oxidation and disulfide formation.
  • Disulfide derivatives (such as a pyridyl disulfide or 5-thio-2-nitrobenzoic acid ⁇ TNB ⁇ derivatives) can also be used to catalyze disulfide bond formation.
  • Functional group A or B in any of the above examples could also be a photoreactive group such as aryl azides, halogenated aryl azides, diazo, benzophenones, alkynes or diazirine derivatives.
  • Reactions of the amine, sulfhydryl, carboxylate groups yield chemical bonds that are described as amide, amidine, disulfide, ethers, esters, isothiourea, isourea, sulfonamide, carbamate, carbon-nitrogen double bond (enamine or imine) alkylamine bond (secondary amine), carbon-nitrogen single bonds in which the carbon contains a hydroxyl group, thio-ether, diol, hydrazone, diazo, or sulfone.
  • Monomers containing vinyl, acrylate, methacrylate, acrylamide, methaacrylamide groups can undergo chain reaction which can be radical, anionic , or cationic. Chain polymerization can also be accomplished by cycle or ring opening polymerization.
  • free radical initiatiors include peroxides, hydroxy peroxides, and azo compounds such as 2,2'-Azobis(-amidinopropane) dihydrochloride ( AAP).
  • a wide variety of monomers can be used in the polymerization processes. These include positive charged organic monomers such as amines, imidine, guanidine, imine, hydroxylamine, hydrozyine, heterocycles (like imidazole, pyridine, morpholine, pyrimidine, or pyrene.
  • the amines could be pH-sensitive in that the pKa of the amine is within the physiologic range of 4 to 8.
  • Specific amines include spermine, spermidine, N,N'-bis(2-aminoethyl)-l,3-propanediamine (AEPD), and 3,3'-Diamino-N,N- dimethyldipropylammonium bromide (Compound 9).
  • Monomers can also be oligopeptides, polypeptides or proteins (produced synthetically or in an organism). These oligopeptides can be a NLS peptide which corresponds to the 12 amino acid nuclear localizing sequence of SV40 T antigen, fusion peptides (derived from viruses), endosomolytic peptides and amphipathic peptides.
  • Amphipathic compounds have both hydrophilic (water-soluble) and hydrophobic (water-insoluble) parts.
  • the amphipathic compound can be cationic or incorporated into a liposome that is positively-charged (cationic) or non- cationic which means neutral, or negatively-charged (anionic). Proteins such as histone HI can be used. Proteins that bind DNA at sequence-specific sequences such as Gal4 protein could also be used.
  • Monomers can also be hydrophobic, hydrophilic or amphipathic.
  • amphipathic compounds include but are not restricted to 3,3'-diamine-N-(7-octene)-N- methyldipropylammonium bromide (Compound 7), N,N'-Dinonacrylate-N,N,N',N'- tetramethylpropanediammonium bromide (Compound 10), N,N',N"-Trinonacrylate- N,N,N',N',N"-pentamethyldiethylentriammonium bromide (Compound 11) and amphipathic peptides such as CKLLKKLLKLWKKLLKKLKC.
  • Monomers can also be intercalating agents such as acridine, thiazole organge, or ethidium bromide.
  • the polymers have other groups that increase their utility. These groups can be incorporated into monomers prior to polymer formation of attached to the polymer after its formation. These groups include:
  • Targeting Groups These groups are used for targeting the polymer-drug or polymer-nucleic acid complexes to specific cells or tissues.
  • targeting agents include agents that target to the asialoglycoprotein receptor by using asiologlycoproteins or galactose residues.
  • Other proteins such as insulin, EGF, or transfemn 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.
  • targeting groups can be used to increase the delivery of the drug or nucleic acid to certain parts of the cell.
  • agents can be used to disrupt endosomes and a nuclear localizing signal (NLS) can be used to target the nucleus.
  • NLS nuclear localizing signal
  • ligands have been used to target drugs and genes to cells and to specific cellular receptors.
  • the ligand may seek a target within the cell membrane, on the cell membrane or near a cell. Binding of ligands to receptors typically initiates endocytosis.
  • Ligands could also be used for DNA delivery that bind to receptors that are not endocytosed. For example peptides containing RGD peptide sequence that bind integrin receptor could be used. In addition viral proteins could be used to bind cells. Lipids and steroids could be used to directly insert into cellular membranes.
  • Reporter Groups are molecules that can be easily detected. Typically they are fluorescent compounds such as fluorescein, rhodamine, texas red, cy 5, or dansyl compounds. They can be molecules that can be detected by UV or visible spectroscopy or by antibody interactions or by electron spin resonance. Biotin is another reporter molecule that can be detected by labeled avidin. Biotin could also be used to attach targeting groups.
  • Cleavable Groups The polymers can contain cleavable groups within the template binding part or between the template binding part and the targeting or reporter molecules. When within the template binding part, breakage of the cleavable groups leads to reduced interaction of the template and daughter polymers. When attached to the targeting group, cleavage leads to reduce interaction between the template and the receptor for the targeting group.
  • Cleavable groups include but are not restricted to disulfide bonds, diols, diazo bonds, ester bonds and sulfone bonds. 5.
  • Template polymerization has been defined as the following (van de Grampel, H.T., Tan, Y. Y. and Challa, G. Macromolecules 23, 5209-5216, 1990):
  • Temporal polymerizations can be defined as polymerizations in which polymer chains are able to grow along template macromolecules for the greater part of their lifetime. Such a mode of propagation can be achieved through the existence of cooperative interactions between the growing chain and the template chain and usually leads to the formation of an interpolymer complex.
  • a well-chosen template is able to affect the rate of polymerization as well as the molecular weight and microstructure of the formed polymer (daughter polymer).
  • the concepts of template polymerization were described by Ballard and Bamford with the ring opening polymerization of the N-carboxyanhydride of DL-phenylalanine on a polysarcosine template.
  • template polymerization propagation of new polymer chain occurs predominantly along the template, a macromolecular chain, through specific cooperative interaction.
  • the nature of interaction can be electrostatic, H- bonding, charge-transfer, and Van der Waals forces in combination with steriochemical matching.
  • the presence of template usually affects various polymerization characteristics as well as the microstructure of the polymer formed.
  • the daughter polymer could be the drug.
  • the template is the drug (defined to include pharmaceuticals, therapeutic agents or biologically active substances).
  • the process of using template polymerization for drug delivery comprises mixing the template with monomers and having a daughter polymer forming from the monomers. The mixture of template polymer and daughter polymer is then administered to a cell by putting the mixture in contact with a cell or near a cell. The mixture of template and daughter polymer could also be placed in a pharmaceutical formulation and vial for delivery to an animal.
  • the template polymer could be a polyanion such as nucleic acid including DNA, RNA or an antisense sequence. The DNA can produce a therapeutic agent such as a therapeutic protein or anti-sense RNA.
  • a polynucleotide can be delivered to block gene expression.
  • Such polynucleotides may be anti-sense by preventing translation of a messenger RNA or could block gene expression by preventing transcription of the gene. Preventing both RNA translation as well as DNA transcription is considered preventing expression. Transcription can be blocked by the polynucleotide binding to the gene as a duplex or triplex. It could also block expression by binding to proteins that are involved in a particular cellular biochemical process.
  • Polynucleotides may be delivered that recombine with genes.
  • the polynucleotides may be DNA, RNA, hybrids and derivatives of natural nucleotides.
  • a polynucleotide is considered in this specification to include a non-natural polynucleotide (not occurring in nature), for example: a derivative of natural nucleotides such as phosphothionates or peptide nucleic acids.
  • Recombine is the mixing of the sequence of a delivered polynucleotide and the genetic code of a gene. Recombine includes changing the sequence of a gene.
  • Polynucleotides may be delivered that are part of viruses and their derivatives or vectors.
  • viruses and their derivative vectors include retrovirus, adenovirus, adenoassociated virus, herpes virus, Sindbis virus, and parvovirus.
  • targeting groups could be added during the initial template polymerization stage or during subsequent polymerization steps.
  • networks or additional networks can be added to the polymer. These could be used to cross-link the polymers.
  • the polymer could be cross-linked to put the template into a "cage".
  • Cross-linking is the linking of two moieties of a polymer to one another using biflinctional chemical linker.
  • Covalent linking biflinctional linkers may be homobifunctional (which involves the same chemical reaction for linking both moieties) or heterobifunctional (involves two different reactions allowing linkage of different functional groups).
  • a cage may be formed around or near the polyion creating a complex of polyion and polymer.
  • Cross-linking the polymer protects the polyion from being destroyed by enzymes and other degrading substrates. For example: If the polyion is DNA, the cross-linked or caging polymer protects DNA from DNases.
  • stable caged polyion particles still bear a net positive charge. However, it is desirable to recharge it so it would interact less with negatively- charged polymers and particles in vivo. Recharging is switching the net polyion particle charge to an opposite charge.
  • Complexes may be formed and continue to function in a solution of changeable tonicity, which means that the solution can be hypotonic, hypertonic or normal tonicity.
  • Hypotonic means any solution which has a lower osmotic pressure than another solution (that is, has a lower concentration of solutes than another solution).
  • a hypotonic solution is the opposite of a hypertonic solution.
  • Normal tonicity in the preferred embodiments is the tonicity of human body fluids, specifically blood.
  • the chemical reaction and polymerization processes can take place in homophase systems in which the monomer and nucleic acid are in the same solution.
  • This solution can be water, alcohol, chloroform, esters, organic solvents, or polar aprotic solvents such as DMF or DMSO or dioxane. They can be mixtures of aqueous and organic solvents.
  • the chemical reaction and polymerization processes can take place in heterophase systems in which the nucleic acid is in one phase and the monomer is in another phase.
  • heterophase systems can be "oil in water” and also “water in oil” where oil is defined as a solvent that has low solubility in water.
  • This approach could enable the formation of micellar-like structures that have the hydrophobic parts of the polynucleotide in the inside of a vesicle and the hydrophilic parts on the outside, or vice-versa.
  • the polymerization reaction can be performed in both direct (oil- in-water) and inverse (water-in-oil) emulsions. This approach allows the use of hydrophobic or amphipathic monomers (Blackley, D.C.
  • Emulsion Polymerization London: Appl. Sci., 1975. Heterophase polymerization enables vesicles, particles, or supramolecular complexes to be produced in which the nucleic acid is on the surface of polymer micelles or the nucleic acid is inside of monolayer inverse polymer micelles. In the last case different lipids can be used for external layer formation. Inverse phase emulsion can be prepared so that in average only one molecules of biopolymer will be present in every water drop.(Martinek, K., Levashov, A.V., Klyachko, N., Khmelnitski, Y.L., & Berezin, I.V. (1986) Eur. J.Biochem. 155, 453-468).
  • Supramolecular Complexes A supramolecular complex is a structure that contains two or more different molecules that are not covalently bound. Supramolecular complexes can be used for drug delivery and for other purposes such as for preparative or analytical methods or the construction of new materials. We describe a new method for forming a supramolecular complex containing nucleic acid and a polymer in which the polymer is formed in the presence of the nucleic acid.
  • the supramolecular complex can contain other components in addition to the nucleic acid and polymer. It can contain another polymer that is already formed. This already formed polymer can bind the nucleic acid or the daughter polymer.
  • the additional component can be a protein. This protein can be cationic and contain positive charges that enables it to bind nucleic acid. Such cationic proteins could be histone, polylysine, or protamine.
  • the supramolecular complex could also contain targeting groups.
  • Liposomes are microscopic vesicles that contain amphipathic molecules that contain both hydrophobic and hydrophilic domains. 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.
  • 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.
  • a method of condensing nucleic acid is defined as decreasing the linear length of the nucleic acid.
  • Condensing nucleic acid also means compacting nucleic acid.
  • Condensing nucleic acid also means decreasing the volume which the nucleic acid molecule occupies.
  • a example of condensing nucleic acid is the condensation of DNA that occurs in cells. The DNA from a human cell is approximately one meter in length but is condensed to fit in a cell nucleus that has a diameter of approximately 10 microns.
  • the cells condense (or compacts) DNA by a series of packaging mechanisms involving the histones and other chromosomal proteins to form nucleosomes and chromatin.
  • the DNA within these structures are rendered partially resistant to nuclease (DNase) action.
  • the condensed structures can also be seen on electron microscopy.
  • the process of condensing nucleic acid can be used for transferring nucleic acids into cells or an organism such as for drug delivery. It could also be used for prepartive or analytical methods or the construction of new materials.
  • a chemical reaction is defined as a molecular change in the participant atoms or molecules involved in the reaction.
  • An example of a molecular change would be the breaking and forming of covalent bonds of participant compounds.
  • Covalent bonds are defined as having shared-electron bonds such as those found in carbon-carbon , carbon-nitrogen, carbon hydrogen, carbon-oxygen, carbon-sulfur, carbon-halogen, nitrogen-hydrogen, oxygen-hydrogen, oxygen-oxygen and sulfur-oxygen bonds.
  • the chemical reaction(s) could result in a polymer being formed.
  • the polymerization process could take place by the process of template polymerization.
  • a supramolecular complex could form as a result of this process.
  • one method utilizes covalently attaching compounds to polyions such as genes for enhancing the cellular transport of the polyion while maintaining its functionality.
  • polyions such as genes for enhancing the cellular transport of the polyion while maintaining its functionality.
  • 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.
  • Nuclear localizing signals enhance the entry of a polyion 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.
  • the process of forming a polymer in the presence of the nucleic acid can be used for transferring nucleic acids into cells or an organism such as for drug delivery. It could also be used for preparative or analytical methods or the construction of new materials.
  • the nucleic acid-binding polymer can form as a result of template polymerization.
  • An interpolyelectrolyte complex is defined as a mixture of two polymers with opposite charges. In this situation the nucleic acid is a polyanion and the formed polymer is a polycation.
  • Orthogonal - refers to a protective (protecting) group that can be selectively removed in the presence of other protective groups contained on the molecule of interest.
  • Monovalent - refers to an ionic species possessing 1 charge.
  • Protective group - A chemical group that is temporarily bound to functionalities within a multifunctional compound that allows selective reactions to take place at other sites within the compound.
  • Common protective groups include, but are not limited to carbamates, amides, and N-alkyl groups.
  • Functionality refers to general classes of organic compounds such as: alcohols, amines, carbonyls, carboxyls, and thiols.
  • polymerization can take place in the presence of DNA. Since the central feature of these polymers is their ability to bind DNA, we selected a relatively simple assay to detect the formation of such polymers and that is agarose gel electrophoresis with ethidium bromide staining of DNA. A strong DNA- binding polymer retards (or slows) the migration of the DNA in the gel. In the experimental samples where the DNA is already present during the polymerization (reaction) process, the sample is simply loaded onto the agarose gel. In the control samples where DNA is not present during the reaction process, the DNA is added after the reaction. This approach is also a powerful method to determine whether any polymer is formed by a template polymerization process.
  • the process of polymerization in the presence of nucleic acids can be used to transfer and express genes in cells. Besides showing the utility of this process, it also indicates that the chemical reactions were not chemically modifying or destroying the nucleic acid.
  • a another approach was also used to detect nucleic acid damage. We incorporated disulfide bonds into the polymers and then broke the polymers down by adding dithiothreitol (DTT also known as Cleland's reagent) which reduces the disulfide bonds. After the breakdown of the polymers the nucleic acid, DNA (that was within the polymer particles) was transfected into cells using another transfection method (with a catiomc lipid). Expression was the same as the native DNA. Expression is a very sensitive indicator of any destruction or modification along the entire length of the reporter (luciferase) gene and promoter. These polymers were designed with disulfide bonds so that they could more easily be broken down inside cells.
  • Step polymerization with DNA as a template was performed using the polyamine N,N'- bis(2-aminoethyl)-l,3-propanediamine (AEPD) and dithiobis(succinimidylpropionate) (DSP).
  • AEPD polyamine N,N'- bis(2-aminoethyl)-l,3-propanediamine
  • DSP dithiobis(succinimidylpropionate)
  • Optimized reaction conditions with AEPD/DSP were as follows. Plasmid DNA (pCIluc, 50 mg) and AEPD (10 mg) were mixed in 50 ml of buffer solution (0.1 M HEPES, 1 mM EDTA, pH 7.4). After 5 min DSP (60 mg in 1.5 ml of dimethylformamide) was added. After mixing, the reaction was left for 1 hour in the dark at room temperature. Finally, reaction mixture was dialysed against water or desired buffer solution in microdialysis cell with a molecular weight cut-off of 1 ,000 (Rainin, Ridgefield, NJ).
  • the pCILuc plasmid expresses a cytoplasmic luciferase from the human immediately early cytomegaloviral (CMV) promoter. It was constructed by inserting the cytoplasmic luciferase cDNA into the pCI (Promega Corp., Madison, WI) CMV expression vector. Specifically, a Nihau/EcoRI restriction digestion fragment containing the cytoplasmic luciferase cDNA was obtained from pSPLuc (Promega Corp.) and inserted into pCI pDNA that was digested with Nhel and EcoRI.
  • CMV immediately early cytomegaloviral
  • Plasmid DNA was purified using the Qiagen (Chatsworth, CA) plasmid purification system (alkaline lysis followed by anion exchange chromatography). Agarose gel electrophoresis and ethidium bromide staining of the DNA was done using standard techniques (Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) in Molecular Cloning Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
  • Standard gel filtration (size-exclusion) chromatography was performed to determine the size of the polymers that formed in the presence and absence of DNA. Since the DNA strongly bound the polymer, it was necessary to first remove the DNA. This was accomplished by vigorous DNase digestion. Samples of DNA/ AEPD/DSP reaction mixture (50 ug total DNA, pCIluc) were supplemented with 5M NaCI solution up to 0.5 M NaCI. DNase I (Sigma) was added to the mixture (0.06 U/ug DNA). DNase digestion was carried out in the buffer containing 10 mM Tris, 10 mM MgC12, 1 mM CaCl2, pH 7.0, for 4 hrs at 37° C.
  • the preparation for light scattering was prepared essentially with the same DNA/AEPD/DSP ratios as for EM (see optimized AEPD/DSP) but with 3 mg of DNA (pCIluc).
  • DNA/AEPD mixture was incubated with occasional vortexing for 10 min at room temperature before addition of DSP.
  • the sample was centrifuged at 12,000 ⁇ m for 5 min and passed through 0.2 um polycarbonate filter (Poretics Corp., Livermore, CA) and analyzed using Particle Size Analyser equipped with 15 M argon laser (Brookhaven Instruments, Inc.).
  • a control sample contained AEPD and DSP at the same concentrations but plasmid DNA was omitted during the reaction. The DNA was added after the reaction was completed. Agarose gel electrophoresis showed much less retardation of the DNA than the above experimental sample. This indicates that polymerization did not occur in the control sample and that the polymerization observed in the experimental sample occurred by template polymerization .
  • the polyamine was co-polymerized with DSP to form a polymer in the presence of DNA and this polymer was bound to the DNA.
  • the polymer condensed the DNA to form particles less than 80 nm in diameter.
  • the DNA was not chemically modified by the polymerization process and was still able to express luciferase after transfection into cells in culture.
  • Step polymerization with DNA as a template was performed using the polyamine N,N'- bis(2-aminoethyl)-l,3-propanediamine (AEPD) as in Example 1 above except DPBP was used as the co-monomer.
  • AEPD polyamine N,N'- bis(2-aminoethyl)-l,3-propanediamine
  • diimidoester cross-linking preserves positive charges of aminogroups by converting them into amidines Therefore, extremely positively charged polymer was formed as a result of this reaction which caused complete DNA retardation on agarose gels DNA addition to the reaction mixture after the reaction between amine and cross-linker did not induce DNA retardation on the gel Treatment of retarded complexes with DTT resulted in restoration of the native plasmid electrophoretic pattern
  • Step polymerization of AEPD and DPBP occurred in the presence of DNA and resulted in a polymer that bound DNA very strongly 2
  • Step polymerization with DNA as a template was performed using the polyamine N,N'- bis(2-aminoethyl)-l,3-propanediamine (AEPD) as in Example 1 above except 2- iminothiolane (Traut's reagent) was used as the co-monomer
  • AEPD polyamine N,N'- bis(2-aminoethyl)-l,3-propanediamine
  • 2- iminothiolane Traut's reagent
  • Optimized reaction conditions with AEPD/2-iminothiolane were as follows. Plasmid DNA (pCILuc, 50 mg), AEPD (1 mM) and iminothiolane (4 mM) were mixed in 450 ml of buffer solution (20 mM HEPES, 1 mM EDTA, pH 8.0). After 30 min 5 ml of iodine solution (40 mM in ethanol) were added. Reaction was allowed to stand for 1.5 h in the dark at room temperature.
  • the above procedure is two-step polymerization with reactive monomer formation.
  • 2-iminothiolane forms bisthiol AEPD derivative on the DNA which can be further polymerized by oxidizing SH groups with molecular iodine.
  • Results for DNA gel retardation and DTT treatment are basically the same as for AEPD/DPBP pair. Under conditions indicated above DNA and AEPD/2-iminothiolane polymer form truly soluble complex completely retarded in agarose gel.
  • the DNA in the control sample (DNA added after the polymer reaction) was not retarded on gel electrophoresis.
  • Ring opening and two-step polymerization processes can be used for forming template polymers that bind to DNA.
  • Polyamines can be polymerized in the presence of DNA using the conversion amines to SH groups with subsequent oxidation reactions.
  • EXAMPLE 4 Similar results were obtained when spermine was used instead of AEPD as in Example 1. Plasmid DNA (10 ug) and spermine (1.5 ug) were mixed in 15 ul of 0.1M HEPES, pH 8.0. After 5 min of incubation DSP (25 ug in 1 ml of DMF) was added with intensive mixing. After 1 hr incubation at room temperature DNA was analyzed on agarose gel. In case of "DNA after" experiment, DNA (10 ug) was added after quenching DSP reaction with 0.1 M glycine for 0.5 hr. Electrophoretic pattern was found similar to the one with AEPD/DSP in Example 1.
  • a novel amine was used as a monomer in conjunction with DTBP for template polymerization of DNA.
  • Optimized reaction conditions with compound 6/DTBP were as follows. Plasmid DNA (pCILacZ, 10 mL of a 3.4 mg/mL stock solution, 34 mg, 103 nmol nucleotide base) and compound 6 (3 mL of a 1.29 mg/mL stock solution, 39 mg, 108 nmol) were mixed with 85 ml water and 10 ml of buffer solution (0.2 M HEPES, 10 mM EDTA, pH 8.0). DTBP (1.1 mL of a 100 mM solution in DMF, 33.7 mg, 109 nmol) was added. After mixing, the reaction was left for 1 h in the dark at room temperature. The pCILacZ plasmid was similarly constructed by placing the restriction digestion fragment of the E. coli ⁇ -galactosidase gene into the pCI vector.
  • a peptide was used as a monomer for polymerization in the presence of DNA and this process enable the formation of complexes that expressed luciferase after transfection into cells in culture.
  • the cross-linkers DSP (dithiobis[succinimidylpropionate]) and DPDPB (1,4- Di-[3'-(2 , -pyridyldthio)-propionamido)]butane) were purchased from Pierce.
  • the NLS peptide was mixed with plasmid DNA (pCILuc) at various ratios (0.4, 0.8, 1.2, 1.6) in 20 mM HEPES pH 7.5, 1 mM EDTA at a concentration of plasmid DNA of 0.3 mg/ml.
  • the disulfide cleavable cross-linker DPDPB was added to final concentrations of 2 and 6 mM and the mixtures were incubated for 30 minutes at room temperature in the dark. Reaction products were analyzed by agarose gel electrophoresis and DNA was visualized by ethidium bromide staining. Extent of polymerization of cationic monomers (NLS peptides) was determined on SDS-PAGE.
  • NLS peptide/pDNA complexes (with and without DPDPB cross-linker) were incubated with 2.5 units DNase I for 1 hour at 37° C to remove the DNA from the complexes. Following digestion, remaining protein components were run on a 15% SDS-PAGE and stained with coomassie blue.
  • NLS peptide/pDNA complexes (with and without DPDPB cross-linker) were diluted in Opti-MEM (Life Technologies) and a flisogenic cationic polyamine (ODAP, Mirus Co ⁇ , Madison, WI) was added to enhance transfection. It is believed that this polyamine helps facilitate intracellular endosomal escape of the complexes into the cytoplasm.
  • Pre-formed complexes were incubated with phosphate buffered saline washed NIH3T3 cells for 4 hours at 37°C. Transfection complexes were then removed and replaced with complete growth medium. Cells were grown for 40 - 48 hours and harvested and assayed for reporter gene expression (luciferase).
  • luciferase activity For determination of luciferase activity, cells were lysed by the addition of 100 ul for 25 mm-in-diameter plates and 200 ul for 35 mm-in-diameter plates of lysis buffer (0.1% Triton X-100, 0.1M K-phosphate, 1 mM DTT pH 7.8). 20 ul of the cellular extract were analyzed for luciferase activity as previously reported (Wolff, J.A., Malone, R.W., Williams, P., Chong, W., Acsadi, G, Jani, A. and Feigner, P.L. Direct gene transfer into mouse muscle in vivo. Science, 1465-1468, 1990.). A Lumat LB 9507 (EG&G Berthold, Bad-Wildbad, Germany) luminometer was used.
  • NLS peptides The stepwise cross-linking of NLS peptides along the DNA template drastically alters the mobility of pDNA in agarose gel electrophoresis.
  • peptide to pDNA ratios 0.4 : 1, 0.8 : 1
  • the NLS peptide/pDNA/DPDPB complexes migrated as a characteristic smear with several prominent bands as compared to NLS peptide/pDNA complexes without cross-linker which migrated similarly to pDNA alone.
  • ratios 1.2 : 1, 1.6 : 1
  • the net charge of the complexes becomes positive and precipitation occurs with or without the DPDPB cross-linker.
  • Lanes M-marker protein standards; 1 - NLS peptide alone (7 ⁇ g); 2 - DNase I alone (2.5 u); 3 - NLS peptide / pDNA; 4 - NLS peptide/DPDPB (2mM) + pDNA added after the polymerization reaction; 5 - NLS peptide/DPDPB (6mM) + pDNA added after the polymerization reaction; 6 - NLS peptide/DPDPB (2mM) / pDNA; 7 - NLS peptide/DPDPB (2mM)/pDNA. Protein staining clearly shows a ladder of increasing size bands indicating a stepwise polymerization of NLS peptides with dimers appearing as the fastest migrating species. This ladder of bands was only observed in the reactions when the cross-linker
  • Peptides can be used for template polymerization in the presence of DNA. 2. This process enables complexes to be prepared that can transfect mammalian cells efficiently.
  • Vim 1 -vinyl imidazole
  • AAP 2,2'-Azobis(-amidinopropane) dihydrochloride
  • plasmid DNA 20mM of plasmid DNA (pBlueRSVLux, 800 ul of 6.9 mg/ml) was mixed with 20 mM of Vim and 2 mM of AAP from the stock solutions above.
  • a control sample contained Vim and AAP at the same concentrations but plasmid DNA was omitted.
  • Both the experimental (VIm/AAP/DNA) and control (Vim/ AAP but no DNA) reactions were performed in sterile deionized water. The reactions were incubated for 2 hours at 50° C and then the samples were analyzed by agarose gel electrophoresis followed by ethidium bromide staining. 20 mM of plasmid DNA was added to the control sample prior to loading it on the gel.
  • plasmid DNA pBlueRSVLux also known as pBS.RSVLux
  • pBS.RSVLux Rous Sarcoma Virus
  • the plasmid also contains the SV40 intron and poly A addition signals for proper and efficient mRNA processing.
  • Polyallylamine (hydrochloride)(55 kDa) (PAA) were obtained from Aldrich.
  • Histone Hl(Type III-S from Calf Thymus) was obtained from Sigma.
  • Dimethyl 3,3'- dithiobispropionimidate(DTBP) was purchased from Pierce.
  • the polycations were dissolved in de ionized water: PLL and HI to concentration lOmg/ml and PAA to 2mg/ml.
  • DTBP was dissolved in H2O (30mg/ml) immediately before utilization.
  • DNA/polycation complexes were prepared by the rapid mixing of 37 ⁇ g of plasmid DNA with varying amounts of polycations in 750 ⁇ l of 25mM HEPES pH 8.0, 0.5 mM EDTA. The mixtures were kept 30 min at room temperature and various amounts of DTBP were added. The mixtures were incubated 2 hours at room temperature. 2M NaCI was added to the complexes to final concentration 100 mM while vigorously mixing.
  • PLL was added to plasmid DNA in 0.75 ml of 25 mM HEPES pH 8.0 while vigorously mixing. The kinetics of light scattering was determined immediately after mixing. The turbidity of DNA PLL complexes was well above that of free DNA in all range of PLL concentration As shown in Fig.4 complex aggregation increased when molar charge ratio PLL/DNA to approximate to land was maximal at ratio 1.17. Further increases in PLL concentration resulted in decreasing of complex turbidity. The light scattering did not change with time for at least for 30 min.
  • Table 3 The effect of varying the DNA/PLL charge (monomoer: monomer) ratio on the sizes of PLL/DNA complexes with the addition of 0.97 umol DTBP.
  • the sizes were determined by quasi elastic light scattering and numbers indicate the percent of particles ⁇ 100 nm or >100 nm. Number in parentheses indicate the size (diameter in nm) of the most abundant species within that size range.
  • PLL/DTBP ratio Effect of PLL/DTBP ratio on the size and stability of PLL/DNA complexes.
  • the PLL/DNA complex in ratio 4.12 was treated by different concentrations of
  • Table 4 The effect of varying the DTBP/PLL ratio (molar ratio of DTBP to lysine residue) on the sizes of PLL/DNA complexes.
  • the sizes were determined by quasi elastic light scattering and numbers indicate the percent of particles ⁇ 100 nm or > 100 nm. Number in parentheses indicate the size (diameter in nm) of the most abundant species within that size range.
  • DSY DNA PLL complexes Stability of DNA PLL complexes to disruption by polyanion dextran sulfate (DSY DNA PLL complexes (molar ratio of 0.87, 1.74, 3.04 or 4.35 as indicated in
  • Fig. 5 were prepared as before but in 1ml of buffer. 0.97 umol of DTBP were added. The mixtures were incubated 2 hours at room temperature. 10 ul of ethidum bromide
  • PAA Polyallylamine
  • HI has total positive charge 55 per molecule (Mw 21.3 kDa) and can form an inter polyelectrolyte complex with DNA.
  • Mw 21.3 kDa total positive charge 55 per molecule
  • interaction of HI with DNA leads to considerable increase of turbidity in broad range of HI concentration.
  • the turbidity is not changed after addition of lOOmM NaCI.
  • Treatment of HI DNA complex with charge ratio 3.42 by DTBP leads to significant decrease of turbidity from 1929 to 348. Following addition of NaCI causes the turbidity to increase to 458.
  • DNA template polymerization of large polymers yields small particles that do not aggregate in physiological salt solutions.
  • the ability to prepare small particles of condensed DNA that do not aggregate in a physiologic salt solution will be an extremely useful formulation for gene transfer and therapy.
  • Step polymerization with DNA as a template was performed using the polyamine N,N'- bis(2-aminoethyl)-l,3-propanediamine (AEPD) as in Example 1 except pegylated AEPD (Compound 18, N2,N2,N3,N3-tetra(PEG-amino propyl) -AEPD, or will be referred to as AEPD-PEG) was added to the reaction mixture along with AEPD.
  • AEPD-PEG pegylated AEPD
  • This example teaches how to prepare non-aggregated, water-soluble particles (diameter ⁇ 100 nm) of condensed DNA via the process of template polymerization.
  • Optimized reaction conditions with AEPD/ AEPD-PEG mixture were as follows. Plasmid DNA (pCIluc, 10 ug), AEPD (58 ug), AEPD-PEG (5 mg) and DTBP (187 ug) were mixed in 0.5 ml of buffer solution (20 mM HEPES, 1 mM EGTA, pH 8.5). Molar ratio of total AEPD (AEPD+AEPD-PEG) to DNA base was 20 : 1. Reaction was allowed to proceed for three hours at room temperature. At 10, 30, 60 and 180 min time points particle sizing was performed using photon correlation spectrometer as described in Example 1. The data obtained was compared with the experiment where only AEPD was used as monomer. Three independent samples of each template polymerization reaction mixture were measured and the data was expressed as mean +/- SD. Transmission electron microscopy of the AEPD/ AEPD-PEG mixture samples was performed as described in Example 1. Results:
  • AEPD/ AEPD-PEG template polymerization mixture Formation of particles of condensed DNA for AEPD/ AEPD-PEG template polymerization mixture was confirmed both by dynamic light scattering and by electron microscopy. Unlike AEPD alone 20: 1 mixture which yields aggregates, AEPD/ AEPD- PEG mixture resulted in increased population of non-aggregated individual DNA particles with the size ⁇ 100 nm of characteristic rod morphology.
  • Template polymerization can be performed in the presence of pegylated AEPD molecules. PEG-containing monomer was included into final condensed DNA complex.
  • Plasmid DNA (pCI luc, 10 mg) in 660 ⁇ L water was combined with 1380 ⁇ L methanol and 660 ⁇ L chloroform containing 144 mg of Compound 11, giving a clear solution with a 7 fold excess of positive charge. The solution was vortexed and allowed to stand at room temperature for 20 minutes. One half of the monophase was reserved. The remaining monophase was separated into two layers by the addition of an additional 375 ⁇ L water. The two resulting layers were separated. The presence of DNA in the chloroform layer was confirmed by absorbence at 260 nm. The sizes of the particles in the chloroform layer and in the reserved monophase were measured on a Brookhaven ZetaPlus particle sizer.
  • the Bligh/Dyer monophase (Bligh, E.G. and Dyer, W.J. (1959) A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol., 37, 911-917.) had two groups of particles in the size range of 80- 128 nm and 7000-10000 nm. The chloroform layer showed one group of particles with a size range of 4-7 nm, however the signal intensity was low.
  • the Bligh Dyer monophase and the chloroform layer were polymerized in the presence of 1% AIBN (Aldrich Chemical Company) for 1 hour at 55 °C. Particle size was measured after the polymerization.
  • the Bligh/Dyer monophase contained one population of particles with a size range of 700-1000 nm.
  • the chloroform layer contained one population of particles with a size range of 340-400 nm.
  • the polymerized reaction products were analyzed on a SDS-PAGE gel (Novex, 10-20% tricene). Approximately 50 ⁇ g of the polymerized reactions and a control consisting of 7 ⁇ g DNA and 50 ⁇ g compound 11 without polymerization were loaded onto the gel and visualized with coumassie staining. A smear of high-molecular weight polymer beginning in the well was observed in both of the polymerization reactions. The control exhibited one band of low molecular weight.
  • Caged DNA particles were prepared as described in example "Template polymerization (caging) of large polymers" with DTBP as cross-linking agent. Zeta-potentials of the obtained particles were measured using Zeta-Plus Photon Correlation Spectrometer (Brookhaven Instruments Co ⁇ .). Ninety degree light scattering measurements and TOTO binding assay were performed using Fluorescence Spectrophotometer. TOTO assay was used to assess the degree of DNA condensation (Wong FM. Reimer DL. Bally MB, Cationic lipid binding to DNA: characterization of complex formation. Biochemistry. 35(18):5756-63, 1996).
  • TOTO assay (Table. 8) demonstrated that DNA stays condensed after formation of negatively charged triple complex though some partial decondensation occured. Caged complex was found more resistant to decondensation during recharging (80% condensation preserved at 400-500 ug DS added). It is possible to recharge DNA PLL complexes with other polyanions. The similar data were obtained with polymethacrylic acid (not shown).
  • H-NMR spectra were recorded on a Bruker AC 250 or a Bruker AC 300 spectrophotometer with chemical shifts given in parts per million downfield from an internal standard of tetramethylsilane.
  • Diamino-N- methyldipropylamine (Aldrich Chemical Co.), Boc anhydride (Aldrich Chemical Co.), triethylamine (Aldrich Chemical Co.), trifluoroacetic anhydride (Aldrich Chemical Co.), 9-bromo-l-nonanol (Aldrich Chemical Co.), acryloyl chloride (Aldrich Chemical Co.), 3-bromopropylamine hydrobromide (Aldrich Chemical Co.), 7-bromo-l-octene (Aldrich Chemical Co.), trimethylamine (25 % solution in water) (Aldrich Chemical Co.), methyl iodide (Aldrich Chemical Co.), N,N,N'N'-t
  • 3,3'-(N',N"-tert-butoxycarbonyl)-N-methyldipropylamine (1) 3,3'-Diamino-N- methyldipropylamine (0.800 mL, 0.721 g, 5.0 mmol) was dissolved in 5.0 mL 2.2 N sodium hydroxide (11 mmol). To the solution was added Boc anhydride (2.50 mL, 2.38 g, 10.9 mmol) with magnetic stirring. The reaction mixture was allowed to stir at room temperature overnight (approximately 18 hours). The reaction mixture was made basic by adding additional 2.2 N NaOH until all t-butyl carboxylic acid was in solution. The solution was then extracted into chloroform (2 x 20 mL).
  • 3,3'-Trifluoroacetamidyl-N-methyldipropylamine (2) 3,3 '-Diamino-N- methyldipropylamine (0.504 mL, 436 mg, 3.0 mmol) and triethylamine (0.920 mL, 6.6 mmol) were dissolved in 20 mL dry methylene chloride. The solution was chilled on an ice bath. Trifluoroacetic anhydride (0.932 mL, 1.39 g, 6.6 mmol) dissolved in 40 ml dry methylene chloride was added dropwise with magnetic stirring over a period of approximately 20 minutes. The reaction was allowed to come to room temperature and to stir overnight (approximately 18 hours). The reaction mixture was washed 2 x
  • 9-Bromononacrylate (3) 9-Bromo-l-nonanol (0.939 g, 4.0 mmol) was dissolved in 4.0 mL anhydrous diethyl ether in a flame dried 10 mL r.b. flask under dry nitrogen. Sodium carbonate (6.36 g, 6.0 mmol) was added to the reaction mixture. Acryloyl chloride (0.356 mL, 0.397 g, 4.2 mmol) dissolved in 3.5 mL anhydrous ether was added dropwise over a period of approximately 10 minutes. The reaction mixture was allowed to come to room temperature and stir for two days.
  • 3-Bromo-l-(trifluoroacetamidyl)propane 4-Bromopropylamine hydrobromide (2.19 g, 10.0 mmol) and triethylamine (1.67 mL, 12.0 mmol) were dissolved in 60 mL dry methylene chloride. The solution was chilled on an ice bath. Trifluoroacetic anhydride (1.69 mL, 2.51 g, 12.0 mmol) dissolved in 60 mL dry methylene chloride was added dropwise over approximately 20 minutes. The reaction was allowed to come to room temperature and was stirred overnight (approximately 18 hours).
  • HH--NNMMRR ((CCDDCCII33)) dd 55..7755 ((mm,, IIHH), 5.00 (m, 2H), 3.60 (m, 2H), 3.45 (s, 9H), 2.05 (m, 2H), 1.75 (m, 2H), 1.40 (m, 6H).
  • N,N'-Dinonacrylate-N,N,N',N'-tetramethylpropanediammonium bromide (10).
  • N,N,N'N'-tetramethylpropane diamine (0.0252 mL, 0.15 mmol) and compound 3 (131 mg, 0.148 mmol) were dissolved in 0.150 mL dimethylformamide.
  • the reaction mixture was incubated at 50 C_ for 5 days.
  • the product was precipitated from the reaction mixture by the addition of ether.
  • N,N',N"-Trinonacrylate-N,N,N',N',N"-pentamethyldiethylentriammonium bromide (11).
  • N,N,N',N',N"-pentamethylethylentriamine (0.031 mL, 0.15 mmol) and compound 3 (187 mg, 0.675 mmol) were dissolved in 0.150 mL dimethylformamide.
  • the reaction mixture was incubated at 50 C_ for 5 days.
  • the product was precipitated from the reaction mixture by the addition of ether.
  • AEPD 275 mg, 1.72 mmol
  • BOC-ON 800 mg, 3.25 mmol, Aldrich Chemical Co.
  • 3 mL tetrahydrofuran was added dropwise with magnetic stirring over approximately 15 minutes.
  • the ice bath was removed and the stirring reaction mixture was allowed to come to room temperature.
  • the solvent was removed on a rotary evaporator, and the residue was dissolved in 20 mL chloroform.
  • the chloroform was washed with 2 N sodium hydroxide.
  • the chloroform layer was then extracted with 0.1 N hydrochloric acid.
  • the precipitate was washed 2x with diethyl ether, and dried under vacuum to yield 198 mg product.
  • the product was dissolved in 2 mL trifluoroacetic acid, and incubated 20 minutes to remove the BOC protecting groups.
  • the trifluoroacetic acid was removed under a stream of nitrogen.
  • the residue was dried under vacuum to yield 198 mg product as an off- white solid.
  • the presence of free amino groups after the removal of the BOC protecting groups was confirmed by a ⁇ positive ninhydrin test.
  • the final product should contain approximately 3 Peg chains per molecule as determined by the amount of NHS-Peg used in reaction.

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Abstract

L'invention concerne un procédé qui permet de former des polymères en présence d'un acide nucléique par une technique de polymérisation matricielle, ainsi qu'une technique qui permet d'effectuer la polymérisation dans des systèmes à plusieurs phases. Ces techniques peuvent être utilisées pour l'apport et la condensation d'acides nucléiques, ainsi que pour la formation de polymères de liaison d'acide nucléique, de complexes supramoléculaires contenant un acide nucléique et un polymère, et de complexes interpolyélectrolytiques.
PCT/US1999/008965 1998-04-30 1999-04-23 Technique permettant de fabriquer un compose en formant un polymere a partir d'un medicament matrice WO1999055825A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP99920014A EP1073707A4 (fr) 1998-04-30 1999-04-23 Technique permettant de fabriquer un compose en formant un polymere a partir d'un medicament matrice

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US7029998A 1998-04-30 1998-04-30
US09/070,299 1998-04-30

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WO1999055825A1 true WO1999055825A1 (fr) 1999-11-04

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Publication number Priority date Publication date Assignee Title
WO2010133975A3 (fr) * 2009-05-22 2011-11-03 Imuthes Limited Polyamino disulfures réductibles tenant lieu de transfectants
US11584924B2 (en) 2016-08-24 2023-02-21 Infusion Tech Method for concentrating microorganism or extracting nucleic acid using DTBP

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US5658728A (en) * 1991-06-19 1997-08-19 Gosney, Jr.; William Milton Templates for nucleic acid molecules
US5741713A (en) * 1995-06-21 1998-04-21 Martek Biosciences Corporation Combinatorial libraries of labeled biochemical compounds and methods for producing same
US5750338A (en) * 1986-10-23 1998-05-12 Amoco Corporation Target and background capture methods with amplification for affinity assays

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US5552309A (en) * 1994-09-30 1996-09-03 Indiana University Foundation Use of polyols for improving the introduction of genetic material into cells
US6126964A (en) * 1996-01-04 2000-10-03 Mirus Corporation Process of making a compound by forming a polymer from a template drug

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US5750338A (en) * 1986-10-23 1998-05-12 Amoco Corporation Target and background capture methods with amplification for affinity assays
US5466589A (en) * 1990-11-19 1995-11-14 Biotechnology Research & Development Corporation Coated substrates employing oriented layers of mutant heme proteins and methods of making and using same
US5310648A (en) * 1991-02-01 1994-05-10 California Institute Of Technology Composition of matter comprising an imprinted matrix exhibiting selective binding interactions through chelated metals
US5658728A (en) * 1991-06-19 1997-08-19 Gosney, Jr.; William Milton Templates for nucleic acid molecules
US5741713A (en) * 1995-06-21 1998-04-21 Martek Biosciences Corporation Combinatorial libraries of labeled biochemical compounds and methods for producing same

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010133975A3 (fr) * 2009-05-22 2011-11-03 Imuthes Limited Polyamino disulfures réductibles tenant lieu de transfectants
US11584924B2 (en) 2016-08-24 2023-02-21 Infusion Tech Method for concentrating microorganism or extracting nucleic acid using DTBP

Also Published As

Publication number Publication date
EP1073707A1 (fr) 2001-02-07
EP1073707A4 (fr) 2002-03-13

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