EP1209971A1 - Charge reversal of polyion complexes - Google Patents
Charge reversal of polyion complexesInfo
- Publication number
- EP1209971A1 EP1209971A1 EP00955747A EP00955747A EP1209971A1 EP 1209971 A1 EP1209971 A1 EP 1209971A1 EP 00955747 A EP00955747 A EP 00955747A EP 00955747 A EP00955747 A EP 00955747A EP 1209971 A1 EP1209971 A1 EP 1209971A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- dna
- complex
- complexes
- polycation
- particles
- 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.)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/58—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
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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
Definitions
- the invention relates to compounds and methods for use in biologic systems. More particularly, polyions are utilized for reversing the charge (“recharging”) particles, such as molecules, polymers, nucleic acids and genes for delivery to cells.
- Polycations provide attachment of DNA to the target cell surface.
- the polymer forms a cross- bridge between the polyanionic nucleic acids and the polyanionic surfaces of the cells.
- Polycations protect DNA in complexes against nuclease degradation. Polycations can also facilitate DNA condensation.
- the volume which one DNA molecule occupies in a complex with polycations is drastically lower than the volume of a free DNA molecule.
- the size of a DNA polymer complex is important for gene delivery in vivo.
- DNA In terms of intravenous injection, DNA must 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.
- 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 and are of similar size in other cell types, DNA complexes smaller than 100 nm are preferred.
- Multivalent cations with a charge of three or higher have been shown to condense DNA. These include spermidine, spermine, Co(NH3)63+ ,Fe3+ , and natural or synthetic polymers such as histone HI, protamine, polylysine, and polyethylenimine. Analysis has shown DNA condensation to be favored when 90% or more of the charges along the sugar-phosphate backbone are neutralized. 2. Polymers (neutral or anionic) which can increase repulsion between DNA and its surroundings have been shown to compact DNA. Most significantly, spontaneous DNA self-assembly and aggregation process have been shown to result from the confinement of large amounts of DNA, due to excluded volume effect.
- Toroids have been considered an attractive form for gene delivery because they have the smallest 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. Therefore one toroid can include from one to several DNA molecules. The kinetics of DNA collapse by polycations that resulted in toroids is very slow For example DNA condensation by Co(NH3)6C13 needs 2 hours at room temperature
- DNA condensation is not clear
- the electrostatic force between unperturbed helices a ⁇ ses primarily from a counte ⁇ on fluctuation mechanism requiring multivalent cations and plays a major role in DNA condensation
- the hydration forces predominate over electrostatic forces when the DNA helices approach closer then a few water diameters
- DNA condensation is a more complicated process than the case of low molecular weight polycations
- Different polycationic proteins can generate toroid and rod formation with different size DNA at a ratio of positive to negative charge of 0 4 T4
- DNA complexes with polyarginine or histone can form two types of structures, an elongated structure with a long axis length of about 350 nm (like free DNA) and dense spherical particles Both forms exist simultaneously in the same solution
- the reason for the co-existence of the two forms can be explained as an uneven distribution of the polycation chains among the DNA molecules The uneven distribution generates two thermodynamically favorable conformations
- the electrophoretic mobility of DNA -polycation complexes can change from negative to positive in excess of polycation It is likely that large polycations don't completely align along DNA but form polymer loops that interact with other DNA molecules The rapid aggregation and strong intermolecular forces between different DNA molecules may prevent the slow adjustment between helices needed to form tightly packed orderly particles
- condensation state of DNA Several methods can be used to determine the condensation state of DNA. They include the prevention of fluorescent molecules such as ethidium bromide from intercalating into the DNA. The condensation state of DNA was monitored as previously described (Dash, RR, Toncheva V, Schacht E, Seymour LW J. Controlled Release 48:269-276) Alternatively the condensation of fluorescein-labeled DNA (or any fluorescent group) causes self-quenching by bringing the fluorescent groups on the DNA closer together (Trubetskoy, VS, Budker, VG, Slattum, PM, Hagstrom, JE and Wolff, JA Analytical Biochemistry 267:309-313, 1999).
- preparation of polycation-condensed DNA particles is of particular importance for gene therapy, more specifically, particle delivery such as the design of non- viral gene transfer vectors
- particle delivery such as the design of non- viral gene transfer vectors
- Optimal transfection activity in vitro and in vivo can require an excess of polycation molecules
- the presence of a large excess of polycations may be toxic to cells and tissues
- the non-specific binding of cationic particles to all cells forestalls cellular targeting. Positive charge also has an adverse influence on biodist ⁇ bution of the complexes in vivo.
- Cationic hp ⁇ d(CL)/DNA complexes can be used as gene delivery vehicles in vitro and in vivo
- hpoplexes Cationic hp ⁇ d(CL)/DNA complexes
- DOTMA N-[l-(2,3-d ⁇ oleyloxy)propyl-N,N,N-t ⁇ methylammon ⁇ um] chloride
- the liver is a relatively large organ and is the secretory source of a large amount of serum proteins
- the liver sinusoidal endothelial fenestrae are ⁇ 150 nm in diameter which essentially allows parenchymal hepatocytes to come in direct contact blood plasma
- These physical and functional characteristics are major factors that rendered liver as an important target tissue for gene therapy
- target cells for gene expression via the vasculature, they must possess certain physical properties a) stability in physiological salt solutions and serum components, b) optimal vector size which is comparable to sinusoidal fenestrae, c) the ability to interact will cell membrane and induce internahzation mechanisms
- the present invention desc ⁇ bes a process for negatively charging DNA particles by recharging fully condensed polycation/DNA complexes with polyions
- a process for delivering a complex to a cell, comprising, forming a compound having a net charge comprising a polyion and a polymer in a solution, adding a charged polymer to the solution in sufficient amount to form the complex having a net charge different from the compound net charge, and, inserting the complex into a mammal
- a complex for delivering a polyion to a cell comprising a polyion and a charged polymer wherein the polyion and the charged polymer are bound in complex, the complex having a net charge that is the same as the net charge of the charged polymer
- a drug for delivery to a cell comprising a polycation non-covalently attached to a polyanion complexed with a negatively charged polyion
- DNA/polycation (PC) complexes lecharged with various polyanions (PA) can be used for gene delivery in viti o and in vivo Precise titration of
- DNA/PC complex with PA results in a significant increase in gene transfer activity both in viti o and in vivo in a narrow range of PA concentrations
- Our method involves the use of PA with high charge density and DNA/CL composition possessing in vivo gene transfer activity
- PA added to DNA/CL results in increased gene transfer activity
- the examples demonstrate that negatively charged DNA containing particles are internalized by hepatocytes via tail vein injections Furthermore, these data indicate that DNA particles that are stable in salt and serum, with sizes comparable to the sinusoidal fenestrae and negatively charged are targetable to and internalized by liver hepatocytes The examples also indicate that negatively charged complexes that are stable in physiological solutions, whether containing DNA or other therapeutic agents, are targetable to liver hepatocytes
- Fig 1 is a scan of mouse hepatocytes showing delivery of cross-linked Cy3-DNA PLL/SPLL particles by tail vein injection H indicates hepatocytes, S indicates sinusoidal Kupffer and endothelial cells
- Fig 2 is a scan of mouse hepatocytes showing delivery of cross-linked Cy3-
- DNA/pAllylamine-cys/pAA-thioester complexes by tail vein injection H indicates hepatocytes
- S indicates sinusoidal cells
- PLL Poly-L-Lysine
- SPLL succinic anhyd ⁇ de-PLL
- pMAA polymethacryhc acid
- pMAA polymethacryhc acid
- Gene therapy research may involve the biological pH gradient that is active within organisms as a factor in delivering a polynucleotide to a cell
- Different pathways that may be affected by the pH gradient include cellular transport mechanisms, endosomal disruption/breakdown, and particle disassembly (release of the DNA)
- Gradients that can be useful in gene therapy research involve ionic gradients that are related to cells For example, both Na+ and K+ have large concentration gradients that exist across the cell membrane Recharging systems can utilize such gradients to influence delivery of a polynucleotide to a cell DNA can be compacted by adding polycations to the mixture By interacting an appropriate cation with a DNA containing system, DNA condensation can take place. Since the ion utilized for compaction may exist in higher concentration outside of the cell membrane compared to inside the cell membrane, this natural ionic gradient can be utilized in delivery systems.
- 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 about 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.
- polymerization can be chain or step. This classification description is more often used that the previous terminology of addition and condensation polymer.
- Step Polymerization In 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
- function B can be (but not restricted to) an lodoacetyl derivative, maleimide, azindine derivative, acryloyl derivative, fluorobenzene derivatives, or disulfide derivative (such as a py ⁇ dyl disulfide or 5- th ⁇ o-2-n ⁇ trobenzo ⁇ c ac ⁇ d ⁇ TNB ⁇ derivatives)
- function B can be (but not restricted to) adiazoacetate or an amine in which a carbodiimide is used
- Other additives may be utilized such as carbonyldiimidazole, dimethylamino py ⁇ dine (DMAP), N- hydroxysuccinimide or alcohol using carbodiimide and DMAP
- function B can be (but not restricted to) an epoxide, oxirane, or an amine in which carbonyldiimidazole or N, N'-disuccinimidyl carbonate, or N- hydroxysuccimmidyl chloroformate or other chloroformates are used If functional group A is an aldehyde or ketone then function B can be (but not restricted to) an hydrazine, hydrazide derivative, amine (to form a Schiff Base that may or may not be reduced by reducing agents such as NaCNBH3 ) or hydroxyl compound to form a ketal or acetal
- function A is a sulfhydryl group then it can be converted to disulfide bonds by oxidizing agents such as iodine (12 ) or NaI04 (sodium pe ⁇ odate), or oxygen (02 )
- Function A can also be an amine that is converted to a sulffiydryl group by reaction with 2- Immothiolate (Traut's reagent) which then undergoes oxidation and disulfide formation
- Disulfide de ⁇ vatives (such as a py ⁇ dyl disulfide or 5-th ⁇ o-2-n ⁇ trobenzo ⁇ c ac ⁇ d ⁇ 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 azide (including halogenated aryl azide), diazo ,
- Reactions of the amine, hydroxyl, sulfhydryl, carboxylate groups yield chemical bonds that are described as amide, amidine, disulfide, ethers, esters, enamine, lmine, urea, isothiourea, isourea, sulfonamide, carbamate, alkylamine bond (secondaryamine), carbon-nitrogen single bonds in which the carbon contains a hydroxyl group, thioether, diol, hydrazone, diazo, or sulfone"
- function B can be (but not restricted to) an hydrazine, hydrazide derivative, amine (to form a Schiff Base that may or may not be reduced by reducing agents such as NaCNBH3) or hydroxyl compound to form a ketal or acetal
- 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 (12) or NaI04 (sodium pe ⁇ odate), or oxygen (02)
- Function A can also be an amine that is converted to a sulfhydryl group by reaction with 2- ⁇ m ⁇ noth ⁇ olate (Traut's reagent) which then undergoes oxidation and disulfide formation
- Disulfide derivatives (such as a py ⁇ dyl disulfide or 5-th ⁇ o-2-n ⁇ trobenzo ⁇ c ac ⁇ d ⁇ 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 diazi ⁇ ne derivatives
- Reactions of the amine, hydroxyl, sulfhydryl, carboxylate groups yield chemical bonds that are described as amide, amidine, disulfide, ethers, esters, enamine, urea, isothiourea, isourea, sulfonamide, carbamate, carbon-nitrogen double bond (lmine), alkylamine bond (secondary amine), carbon-nitrogen single bonds in which the carbon contains a hydroxyl group, thio- ether, diol, hydrazone, diazo, or sulfone
- Chain Polymerization In chain-reaction polymerization growth of the polymer occurs by successive addition of monomer units to limited number of growing chains The initiation and propagation mechanisms are different and there is usually a chain-terminating step The polymerization rate remains constant until the monomer is depleted
- 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
- chain reaction which can be radical, anionic , or cationic Chain polymerization can also be accomplished by cycle or ring opening polymerization
- free radical mitiatiors include peroxides, hydroxy peroxides, and azo compounds such as 2,2'-Azob ⁇ s(-am ⁇ d ⁇ nopropane) dihydrochlo ⁇ de ( AAP)
- a compound is a material made up of two or more elements
- Monomers can be used in the polymerization processes These include positive charged organic monomers such as amines, lmidine, guamdine, lmine, hydroxylamine, hydrozyine, heterocycles (like lmidazole, py ⁇ dine, mo ⁇ hohne, py ⁇ midine, 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'-b ⁇ s(2-am ⁇ noethyl)-l ,3-propaned ⁇ am ⁇ nc (AEPD), and 3,3'-D ⁇ am ⁇ no-N,N- dimethyldipropylammonium bromide
- Monomers can also be hydrophobic, hydrophilic or amphipathic
- 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, ohgonucleotides 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 Monomers can also be intercalating agents such as acndine, thiazole organge, or ethidium bromide
- Lipids are amphipathic compounds which are a fat Fat is a glyceryl ester of fatty acids
- Fatty acids is a term that is used to describe the group of substances which are soluble in hydrocarbons and insoluble in water They may be saturated or unsaturated
- the polymers have other groups that increase their utility These groups can be mco ⁇ orated into monomers prior to polymer formation or attached to the polymer after its formation These groups include Targeting Groups- such groups are used for targeting the polymer-nucleic acid complexes to specific cells or tissues
- Targeting Groups- such groups are used for targeting the 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 transfer ⁇ n can be used for targeting Protein refers to a molecule made up of 2 or more ammo acid residues connected one to another as in a polypeptide
- the ammo acids may be naturally occurring or synthetic Peptides that include the RGD sequence can be used to target many cells
- Peptide refers to a linear series of ammo acid residues connected to one another by peptide bonds between the alpha-amino group and carboxyl group
- 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
- hgands have been used to target drugs and genes to cells and to specific cellular receptors
- the hgand may seek a target within the cell membrane, on the cell membrane or near a cell
- Binding of hgands to receptors typically initiates endocytosis
- Ligands could also be used for DNA delivery that bind to receptors that are not endocytosed
- peptides containing RGD peptide sequence that bind integ ⁇ n receptor could be used
- viral proteins could be used to bind the complex to cells
- Lipids and steroids could be used to directly insert a complex into cellular membranes
- the polymers can also contain cleavable groups within themselves When attached to the targeting group, cleavage leads to reduce interaction between the complex and the receptor for the targeting group
- Cleavable groups include but are not restricted to disulfide bonds, diols, diazo bonds, ester bonds, sulfone bonds, acetals, ketals, enol ethers, enol esters, enamines and lmines
- Reporter or marker molecules are compounds that can be easily detected Typically they are fluorescent compounds such as fluorescein, rhodamine, texas red, CY-5, CY-3 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
- 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-polymeric molecule that 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-polymeric molecule that contains two or more negative charges
- the term polyion includes polycation, polyanion, zwitte ⁇ onic polymers, and neutral polymers
- the term zwitte ⁇ onic refers to the product (salt) of the reaction between an acidic group and a basic group that are part of the same molecule Salts are ionic compounds
- a chemical reaction can be used to attach a signal to a nucleic acid complex.
- the signal is defined in this specification as a molecule that modifies the nucleic acid complex and can direct it to a cell location (such as tissue cells) or location in a cell (such as the nucleus) either in culture or in a whole organism.
- the signal can be a protein, peptide, lipid, steroid, sugar, carbohydrate, nucleic acid or synthetic compound.
- the 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 or particle 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
- ligands that enhance endocytosis by enhancing binding the cell surface
- Other proteins such as insulin, EGF, or transfernn 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 amphote ⁇ cin denvatives
- viral proteins could be used to bind cells
- the present invention provides compounds used in systems for the transfer of polynucleotides, o gonucleotides, and other compounds into association with cells within tissues in situ and in vivo
- the process of delivering a polynucleotide to a cell has been commonly termed “transfection” or the process of “transfectmg” 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 and research pu ⁇ oses 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 More particularly, biologically active compounds utilized in this specification are designed to change the natural processes associated with a living cell
- a cellular natural process is a process that is associated with a cell before delivery of a biologically active compound
- the cellular production of, or inhibition of a material, such as a protein, caused by a human assisting a molecule to an in vivo cell is an example of a delivered biologically active compound
- Pharmaceuticals, proteins, peptides, polypeptides, hormones, cytokines, antigens, viruses, ohgonucleotides, and nucleic acids are examples of biologically active compounds
- nucleic acid is a term of art that refers to a polymer containing at least two nucleotides
- Nucleotides contain a sugar deoxy ⁇ bose (DNA) or ⁇ bose (RNA), a base, and a phosphate group Nucleotides are linked together through the phosphate groups
- Bases include pu ⁇ nes and py ⁇ midines, which further include natural compounds adenine, thymme, guanine, cytosine, uracil, inosine, and synthetic derivatives of pu ⁇ nes and py ⁇ midines, or natural analogs
- Nucleotides are the monome ⁇ c units of nucleic acid polymers
- a "polynucleotide” is distinguished here from an "ohgonucleotide” by containing more than 80 monome ⁇ c units, ohgonucleotides contain from 2 to 80 nucleotides
- nucleic acid includes deoxy ⁇ bonucleic acid (DNA
- transfection reagent is a compound or compounds used in the prior art that b ⁇ nd(s) to or complex(es) with ohgonucleotides and polynucleotides, and mediates their entry into cells
- the transfection reagent also mediates the binding and internahzation of ohgonucleotides and polynucleotides into cells
- transfection reagents include cationic hposomes and lipids, polyamines, calcium phosphate precipitates, histone proteins, polyethylenimine, and polylysine complexes.
- the transfection reagent has a net positive charge that binds to the ohgonucleotide 's or polynucleotide's negative charge.
- the transfection reagent mediates binding of ohgonucleotides and polynucleotides to cells 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 or RNA.
- Polyethylenimine which facilitates gene expression without additional treatments, probably disrupts endosomal function itself.
- 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 “electroporafion,” in which a device is used to give an electric charge to cells. The charge increases the permeability of the cell.
- Ionic (electrostatic) interactions are the non-covalent association of two or more substances due to attractive forces between positive and negative charges, or partial positive and partial negative charges.
- Condensed Nucleic Acids Condensing a polymer means decreasing the volume that the polymer occupies.
- An 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 is rendered partially resistant to nuclease DNase) action.
- the process of condensing polymers can be used for delivering them into cells of an organism.
- a delivered polymer can stay within the cytoplasm or nucleus apart from the endogenous genetic material.
- the polymer could recombine (become a part of) the endogenous genetic mate ⁇ al
- DNA can insert mto chromosomal DNA by either homologous or non- homologous recombination
- Condensed nucleic acids may be delivered lntravasculary, lntrarte ⁇ ally, intravenously, orally, intraduodenaly, via the jejunum (or lleum or colon), rectally, transdermally, subcutaneously, intramuscularly, intrape ⁇ toneally, intraparenterally, via direct injections into tissues such as the liver, lung, heart, muscle, spleen, pancreas, brain (including lntravent ⁇ cular), 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 exocnne glands "Delivered" means that the polynucleotide becomes associated with the cell The polynucleotide can
- Intravascular means within a tubular structure called a vessel that is connected to a tissue or organ within the body within the cavity of the tubular structure, a bodily fluid flows to or from the body part
- bodily fluid include blood, lymphatic fluid, or bile
- vessels include arteries, arte ⁇ oles, capillaries, venules, sinusoids, veins, lymphatics, and bile ducts
- the intravascular route includes delivery through the blood vessels such as an artery or a vein
- An administration route involving the mucosal membranes is meant to include nasal, bronchial, inhalation into the lungs, or via the eyes
- Polyions for gene therapy and gene therapy research can involve anionic systems as well as charge neutral or charge-positive systems
- the ionic polymer can be utilized in "recharging" (another layer having a different charge) the condensed polynucleotide complex
- the resulting recharged complex can be formed with an appropriate amount of charge such that the resulting complex has a net negative, positive or neutral charge
- the interaction between the polycation and the polyanion can be ionic, can involve the ionic interaction of the two polymer layers with shared cations, or can be crosslinked between cationic and anionic sites with a crosslinking system (including cleavable crosslinking systems, such as those containing disulfide bonds).
- the interaction between the charges located on the two polymer layers can be influenced with the use of added ions to the system.
- the layers can be made to disassociate from one another as the ion diffuses from the complex into the cell in which the concentration of the ion is low (use of an ion gradient).
- Electrostatic complexes between water-soluble polyelectrolytes have been studied widely in recenty ears. Complexes containing DNA as a polyanionic constituent only recently came to the attention because of their potential use in gene therapy applications such as non-viral gene transfer preparations (polyplexes) for particle delivery to a cell. Strong polyelectrolytes, polyanion polycation complexes, are usually formed at a 1 : 1 charge stoichiometrically. A charge ratio 1 : 1 complex between DNA and Poly-L-Lysine (PLL) also has been demonstrated in the prior art.
- PLL Poly-L-Lysine
- Polyanions effectively enhance the gene delivery / gene expression capabilities of all major classes of polycation gene delivery reagents.
- SPLL succinic anhydride- PLL
- SPLL is added to a cationic nucleic acid/PLL complex in solution.
- Nucleic acid at the core of such complexes remains condensed, in the form of particles ⁇ 50 nm in diameter.
- DNA and PLL binds SPLL in 1 : 1 : 1 complex with SPLL providing a net negative charge to the entire complex.
- SPLL succinic anhydride- PLL
- a wide a variety of polyanions can be used to recharge the DNA/polycation particles. They include (but not restricted to): Any water-soluble polyanion can be used for recharging pu ⁇ oses including succinylated PLL, succinylated PEI (branched), polyglutamic acid, polyaspartic acid, polyacryhc acid, polymethacryhc acid, polyethylacryhc acid, polypropylacryhc acid, polybutylacryhc acid, polymaleic acid, dextran sulfate, heparin, hyaluronic acid, polysulfates, polysulfonates, polyvinyl phosphoric acid, polyvinyl phosphomc acid, copolymers of polymaleic acid, polyhydroxybuty ⁇ c acid, acidic polycarbohydrates, DNA, RNA, negatively charged proteins, pegylated derivatives of above polyanions, pegylated derivatives carrying specific ligands, block and
- the recharged nucleic acid complexes (polyanion /polycation/nucleic acid complex) are formed in a container and then administered to the cell or organism
- the polycation nucleic acid complex is recharged with a polyion prior to delivery to the organism and the nucleic acid remains condensed
- the nucleic acid can remain more than 50%, 60%, 70%, 80%, 90% or 100% condensed as well
- DNA forms soluble condensed (toroid) structures stabilized with an excess of polyion
- a third polyelectrolyte is present, a tertiary complex exists In the absence of salt such tertiary complex might exist indefinitely If the last added polyion is in excess, it stabilizes the complex in the form of a soluble colloid Using this method, a DNA/polycation complex, which maintains a net positive charge, reverses its charge and becomes "recharged"
- the complex can be designed (e g choice of polycation and polyanion, presence of crosslinking) so that in the presence of salt, the complex dissociates into binary complex and free excess of third polyion
- tertiary DNA PLL/SPLL complex exhibit the same colloid properties as binary DNA PLL complex In low salt solution it forms flocculate around PLL/SPLL charge equivalence point (FIG 1 )
- Polyanions with high charge density polymethacryhc acid, pMAA and polyaspartic acid, pAsp
- polyglutamic acid, pGlu, SPLL failed to decondense DNA
- FIG 3 Together with z-potential measurements (FIG 3), these data represent support for the presence of negatively charged condensed DNA particles These particles are approximately 50 nm in diameter in low salt buffer as measured by atomic force microscopy (FIG 2) which revealed particles of spheroid mo ⁇ h
- the polyanion can be covalently attached to the polycation using a variety of chemical reactions without the use of crosshnker
- the polyanion can contain reactive groups that covalently attach to groups on the polycation The types of reactions are similar to those discussed above in the section on step polymerization
- the attachment of the recharged complex can be enhanced by using chelators and crown ethers, preferably polyme ⁇ c
- the DNA polycation complexes are initially formed by adding only a small excess of polycation to nucleic acid (in charge ratio which is defined as ratio of polycation total charge to polyanion total charge at given pH)
- charge ratio which is defined as ratio of polycation total charge to polyanion total charge at given pH
- the charge ratio of polycation to nucleic acid charge could be less than 2, less than 1.7, less than 1.5 or even less than 1.3. This would be preferably done in low ionic strength solution so as to avoid the complexes from flocculation.
- Low ionic strength solution means solution with total monovalent salt concentration less than 50 mM Then the polyanion is added to the mixture and only a small amount of "blank” particles are formed "Blank” particles are particles that contain only polycation and polyanion and no nucleic acid.
- the polycation is added to the nucleic acid in charge excess but the excess polycation that is not in complex with the nuclei acid is removed by punficaton.
- Purification means removing of charged polymer using centrifugation, dialysis, chromatography, electrophoresis, precipitation, extraction
- a ultracentnfugation procedure (termed “centrifugation step") is used to reduce the amount of excess polycation, polyanion, or "blank” particles.
- the method is based on the phenomenon that only dense DNA-containing particles can be cent ⁇ fuged through 10% sucrose solution at 25,000g After centrifugation purified complex is at the bottom of the tube while excess of polyanion and "blank” particles stay on top.
- 40% solution of metnzamide can be used as a cushion to collect purified DNA polycation/polyamon complex on the boundary for easy retrieval.
- the attachment of the polyanion to the DNA polycation complex enhance stability but can also enable a hgand or signal to be attached to the DNA particle. This is accomplished by attaching the hgand or signal to the polyanion which in turn is attached to the DNA particle
- a dialysis step or centifugation step can be used to reduce the amount of free polyanion containing a hgand or signal that is in solution and not complexed with the DNA particle
- One approach is to replace the free, uncomplexed polyanion containing a hgand or signal with free polyanion that does not contain a hgand or signal
- a polyanion used for charge reversal is modified with neutral hydrophilic polymer for ste ⁇ c stabilization of the whole complex
- the complex formation of DNA with pegylated polycations results in substantial stabilization of the complexes towards salt- and serum-induced flocculation (Wolfert et al. Hum Gene Therapy 7 2123-2133 (1996), Ogns et al , Gene Therapy 6-595-605 (1999)
- modification of polyanion in t ⁇ ple complex also significantly enhances salt and serum stability
- a polyanion used for charge reversal is cleavable
- cleavable polyion 1 A polyion cleavable in backbone
- First scenario would comprise monomers linked by labile bonds such as disulfide, diols, diazo, ester, sulfone, acetal, ketal, enol ether, enol ester, lmine and enamine bonds
- Second scenario would involve reactive groups (I e electrophiles and nucleophiles) in close proximity so that reaction between them is rapid Examples include having corboxyhc acid derivatives (acids, esters and amides) and alcohols, thiols, carboxylic acids or amines in the same molecule reacting together to make esters, thiol esters, anhydrides or amides
- the polyion contains an ester acid such as citraconnic acid, or di
- Cleavable means that a chemical bond between atoms is broken
- Labile also means that a chemical bond between atoms is breakable
- 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 homobi functional molecule, or different as in a heterobifucnctional molecule
- Example 1 Materials Plasmid DNA ( pCILuc) used for the condensation studies was provided by Bayou Biolabs, Harahan, LA Poly-L-lysine (PLL) (MW 34 kDa), poly-L-aspartic acid (PAA) (MW 36 kDa), poly-L-glutamic acid (PLG) (MW 49 kDa) and rhodamine B isothiocyanate were products of Sigma (St Louis, MO) Polymethacryhc acid (PMA), metnzamide and fluoresceine isothiocyanate were from Ald ⁇ ch (Milwaukee, WI) LabellT kits (Minis Co ⁇ , Madison, WI) were used for covalent labeling DNA with fluorescein and rhodamine Synthesis of succinylated PLL (SPLL).
- PLL Poly-L-lysine
- PAA poly-L-aspartic acid
- PLAG poly-L-glutamic acid
- DNA polyion complex formation DNA/PLL/SPLL complexes were formed in 25 mM
- DNA-containing complexes were retrieved from sucrose/metrizamide boundary using Pasteur pipet and were dissolved in 2.5 M NaCl solution. Visible spectra of the complexes and 1 : 1 premixed Rh-DNA Fl-PLL and Rh-DNA Fl-SPLL standards (700 - 400 nm) were recorded using Shimadzu UV 1601 spectrophotometer.
- Example 2 Recharging of Polyion Condensed DNA Particles The chief DNA polycation complex used was DNA PLL (1:3 charge ratio) formed in low salt buffer. At these conditions, plasmid DNA is completely condensed and compacted into toroid-shaped soluble particles stabilized with excess of polyion (Kabanov et al. Adv. Drug Delivery Rev 30:49-60 (1998). The DNA particles were characterized after addition of a third polyion component to such binary DNA polyion complex. It has been shown that polyanion (polymer or negatively-charged lipid bilayer) can release DNA from its complex with cationic liposomes.
- the aggregation state of condensed DNA particles was determined using both static and dynamic light scattering techniques.
- turbidity of the reaction mixture an indication of aggregation, increases when the lysine to lysyl succinate (NH2/COOH) ratio approaches 1 : 1 (FIG. 1(B)).
- NH2/COOH lysine to lysyl succinate
- FIG. 1(C) demonstrates the change of particle surface charge (zeta potential) during titration of DNA/PLL (1:3) particles with SPLL. The particle becomes negatively charged and accordingly recharged at approximately the equivalence point (FIG. 1(C)).
- FIG. 2 shows small and non-aggregated 50 nm DNA PLL/SPLL spheroids adsorbed on mica in the presence of 1 mM NiC12.
- Any water-soluble polyanion can be used for recharging pu ⁇ oses including succinylated PLL, succinylated PEI, polyglutamic acid, polyaspartic acid, polyacrylic acid, polymethacryhc acid, dextran sulfate, heparin, hyaluronic acid, DNA, RNA, negatively charged proteins, polyanions graft- copolymerized with hydrophilic polymer, and the same carrying specific ligands.
- succinylated PLL succinylated PEI
- polyglutamic acid polyaspartic acid
- polyacrylic acid polymethacryhc acid
- dextran sulfate polymethacryhc acid
- dextran sulfate dextran sulfate
- heparin heparin
- hyaluronic acid DNA
- DNA RNA
- negatively charged proteins negatively charged proteins
- Rh-DNA Fl-PLL/SPLL and Rh-DNA/PLL/Fl-SPLL complexes were formed in low salt buffer and then separated from non-bound polyelectrolyte using density gradient ultracentrifugation. Corresponding amounts of each constituent can be determined by measuring optical density at 495 nm and 595 mn respectively. DNA complexes sediment through 10% sucrose solution and are retained in the separating layer between 10% sucrose and 40% metrizamide (metrizamide cushion).
- Rh-DNA Fl-PLL/SPLL Rh-DNA Fl-PLL/SPLL
- Rh-DNA/PLL/Fl-SPLL FIG. 3b
- Example 4 Zeta Potential of Purified Particles: As one may conclude from stoichiometry studies, the DNA PLL/SPLL (1 :3: 10) initial mixture along with 7x excess of free SPLL also contains 2x excess of PLL/SPLL particles ("blank particles") not complexing DNA. These particles were found not to enter the 10% sucrose layer ensuring complete separation of DNA containing particles from PLL and SPLL excess. Zeta potential was measured using Brookhaven Instruments Co ⁇ . Zeta Plus Zeta Potential Analyzer. DNA concentration was 20 mg/ml in 1.5 ml of 25 mM HEPES, pH 7.5.
- Recharged DNA PEI complexes have reduced toxicity and exhibit gene transfer activity in vivo in an organism. Recharging of DNA polycation complexes with strong polyanions which help to release DNA can also make complexes less toxic in vivo. Resulting complexes also are active in gene transfer in lungs upon i/v administration in mice. Table 1 shows the toxicity of DNA PEI/dextran sulfate (DS) complex is decreasing with the increase of DS content. Tertiary DNA PEI/dextran sulfate complexes were formed in 290 mM glucose, 5 mM HEPES, pH 7.4 at DNA concentration of 0.2 mg/ml and PEI concentration of 0.4 mg/ml.
- Example 7 Crosslinking of polycation and polyanion layers on the DNA-containing particles increases their stability in serum and on the cell surface.
- Negatively charged (recharged) particles of condensed DNA can possess the same physico- chemical properties as positively charged (non-recharged) ones. This includes flocculation in high salt solutions (including physiologic concentration).
- chemical cross- linking of cationic and anionic layers of the DNA particles can substantially improve stability of the particles in serum as well as on the cell surface.
- Table 2 shows the time course of unimodal particle size of DNA/PLL/SPLL crosshnked and non-crosshnked particles in 80% bovine serum as determined by dynamic light scattenng
- Crosshnked particles essentially do not change their size in 200 min at room temperature while non-crosshnked control flocculates rapidly.
- Crosslinking with cleavable reagents might help to overcome an inactivity problem
- the polymers can also contain cleavable groups within themselves When attached to the targeting group, cleavage leads to reduce interaction between the complex and the receptor for the targeting group.
- Cleavable groups include but are not restricted to disulfide bonds, diols, diazo bonds, ester bonds, sulfone bonds, acetals, ketals, enol ethers, enol esters, enamines and lmines, acyl hydrazones, and Schiff bases
- DNA-containing particles were prepared using the procedure in Example 1 with the exception that SPLL-PEG conjugate was doubled compared to SPLL
- Table 3 shows the time course of unimodal particle size of DNA/PLL/SPLL and DNA PLL/PEG-SPLL particles in 80% bovine serum as determined by dynamic light scattering Pegylated particles exhibit higher stability towards flocculation as opposed to non- pegylated ones
- the graph shows in vitro transgene activity of DN A Lipofectamine complexes recharged with polyglutamic acid.
- Example 10 Enhancement of in vitro transgene activity of DNA/LTl (Minis Co ⁇ ., 3:2 DNA lipid ratio, w/w) complexes with PA treatment. Complexes were formed as specified in Example 1.
- the graph shows in vitro transgene activity of DNA/LT 1 complexes recharged with polymethacryhc acid and dextran sulfate.
- Example 12 Use of DNA/DOTAP:cholesterol complexes recharged with PAA for enhancement of gene delivery in lung.
- Mice were injected via tail vein with 250 1 of PAA recharged complexes (50 ⁇ g DNA/animal). Lungs were harvested and homogenized at 4oC after 24 hrs. Luciferase activity of extracts ( 10 ⁇ L) was measured using a Lumat LB 9507 luminometer (EG&G Berthold).
- Fig. 1 shows the enhancement of transgene activity in lungs upon addition of PAA. Complete flocculation of the sample occurred in the range of 30-50 ⁇ g of PAA added. The data demonstrates almost 2 orders of magnitude increase on transgene activity in lungs after recharging DNA CL complexes with strong polyanion and essentially no activity past flocculation point.
- the graph shows transgene activity in lung after i/v administration of DNA/DOTAP cholesterol/PAA complexes
- Example 13 Hepatocytes delivery of cross-linked tertiary DNA/PLL/SPLL complexes by tail vein injection
- Plasmid DNA (pCILuc) were labeled with Cy3 LableIT( Minis Co ⁇ oration, Madison WI) Labeled DNA were typically dissolved in water at concentrations ranged from 1 5-2 mg/ml Poly-L-Lysine, PLL (MW 31 kDa), dissolved in water at 10 mg/ml was purchased from Sigma Chemicals ( St Louis, MO) Succinylated PLL (SPLL) was prepared as previously described and dissolved in water at 20 mg/ml DNA PLL/SPLL cross-linked tertiary complexes were formed at a charge ratio of 1 :3: 10 as follows for a single injection:
- SPLL (345 ug in 50 ul of 20mM MES, pH 5) were activated with the addition of 292 ug of EDC followed by 583 ug of sulfo-NHS, both were dissolved in rfcO at 100 mg/1.2 ml, and incubated for 10 min.
- 50 ug of cy3-labeled DNA in 100 ul of 20 mM MES, pH 6.5 was added to 95 ug of PLL in 100 ul of 20 mM MES, pH 6.5 and mixed immediately.
- the condensed DNA/PLL complexes were added immediately to the activated SPLL solution and mixed thoroughly.
- the cross-linked particles were allowed to incubate at room temperature for at least 2 hr before in-vivo injections. Typically, majority of the particles size ranged from 60-200 nm with an average size around 130 nm and a Zeta- potential of - 40 mV. Salt and serum stability of particles were evaluated by particles size changes over time in the presence of physiologic salt solution or serum.
- the cross-linked particles solution containing 50 ug of Cy3-DNA in 250 ul were injected into a mouse through the tail vein. After 3 hrs, the animal was sacrificed, liver samples were submerged in HistoPrep (Fisher Scientific) and snapped frozen in liquid nitrogen. Frozen liver sections, 4-5 um thick, were prepared and were counter stained sequentially for 20 min each by 10 nm Sytox green ( Molecular Probe) in PBS for cell nuclei and 15 ng/ml of Alexa 488 phalloidin (Molecular Probe) in PBS for actin filaments. Stained slides were analyzed for hepatocytes uptake of Cy3-DNA containing particles using a Zeiss laser scanning confocal microscope.
- Figure 1 shows the fluorescence signals from 10 consecutive confocal planes superimposed to form one image, each plane was 0.45 um thick. With the average size of a mouse hepatocyte around 25-30 um thick, the composite image roughly represent 1/4 of total signals per hepatocytes. It showed that each cell contained 20-40 punctate signals. Each punctate signal may represent endosomes at various stages of the pathway and may contain one or more DNA containing particles. Hepatocytes were distinguishable by their larger size in comparison to other cells and bi-nucleated for a large percentage of the population. A few of the hepatocytes were indicated by (H). A large number of particles were also found in Kuppfer and endothelial cells.
- succinimidyl ester was then added, with rapid stirring, to a solution of polyallylamine hydrochloride MW 50,000 (10 mg, 0.8 eq) that had been dissolved in a solution of methanol (20 mL) and diisopropylethylamine (0.5 mL). After one hour, the solvents were removed by rotary evaporation. The white solid was then dissolved in trifluoroacetic acid (5 mL), triisopropylsilane (0.25 mL), and water (0.25 mL). The two hours, the solvents were removed by rotary evaporation. The resulting solid was then dissolved in water (25 mL) and the pH was adjusted to 9 by the addition of potassium carbonate.
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EP1259265B1 (en) | 2000-03-03 | 2011-06-01 | Genetronics, Inc. | Nucleic acid formulations for gene delivery |
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WO2005032597A1 (en) | 2003-09-29 | 2005-04-14 | Nitto Denko Corporation | Biodegradable polyacetals for in vivo polynucleotide delivery |
US7674452B2 (en) | 2005-03-16 | 2010-03-09 | Nitto Denko Corporation | Polymer coating of cells |
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ROSS PC ET AL.: "Lipoplex size is a major determinant of in vitro lipofection efficiency" GENE THERAPY, vol. 6, no. 4, April 1999 (1999-04), pages 651-659, XP002270219 * |
See also references of WO0113723A1 * |
VITIELLO ET AL.: "Condensation of plasmid DNA with polylysine improves liposome-mediated gene transfer into established and primary muscle cells" GENE THERAPY, vol. 3, 1996, pages 396-404, XP009025639 * |
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