WO2012094574A2 - Nanoparticules de polyribonucléotide stabilisées - Google Patents

Nanoparticules de polyribonucléotide stabilisées Download PDF

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WO2012094574A2
WO2012094574A2 PCT/US2012/020445 US2012020445W WO2012094574A2 WO 2012094574 A2 WO2012094574 A2 WO 2012094574A2 US 2012020445 W US2012020445 W US 2012020445W WO 2012094574 A2 WO2012094574 A2 WO 2012094574A2
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nanoparticles
polycationic
polymeric nanoparticles
cells
sirna
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PCT/US2012/020445
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WO2012094574A3 (fr
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Hai-Quan Mao
Masataka Nakanishi
Rajesh Patil
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The Johns Hopkins University
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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
    • 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
    • A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6935—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • siRNA Small interfering RNA
  • RNAi RNA interference
  • siRNA carriers based on various cationic polymers, lipids, and peptides, have been used to form nanosized complexes with siRNA.
  • these polycation/siRNA nanoparticles still exhibit poor stability in buffers at physiological ionic strength, or in serum-containing media, due to the small molecular weight of siRNA chains.
  • micellar nanoparticles with a complex core surrounded by a PEG corona.
  • the advantages of these micellar nanoparticles include smaller and more uniform size, improved colloidal stability in serum-containing media, higher protection of incorporated DNA against enzymatic degradation, and prolonged blood circulation.
  • PEG-A-PPA condensed micelles with siRNA suffered from low stability in salt solution, which may be due to the short and rigid structure of siRNA in contrast to plasmid DNA.
  • nanoparticles which have improved stability in salt and serum-containing solutions, and that have the ability to protect incorporated nucleic acids such as siRNA against enzymatic degradation in blood and other bodily fluids, while also having high efficiency in delivering the nucleic acids to the target cytosol.
  • the present invention provides one or more polymeric nanoparticles comprising: (a) one or more polycationic polymers, (b) at least one or more polyribonucleotide molecules, and (c) an anionic stabilization reagent.
  • the present invention provides one or more polymeric nanoparticles comprising: (a) one or more polycationic polymers selected from the group consisting of linear or branched polycationic homopolymers, block polycationic copolymers and graft polycationic copolymers, (b) at least one or more polyribonucleotide molecules, and (c) an anionic stabilization reagent.
  • the present invention provides one or more polymeric nanoparticles comprising: (a) one or more polycationic polymers selected from the group consisting of linear and/or branched polyethyleneimines (PEI), polyphosphoroamidates (PPA), block polycationic copolymers comprising PEG and PEI or PPA, and graft polycationic copolymers comprising PEG and PEI or PPA, (b) at least one or more polyribonucleotide molecules, and (c) an anionic stabilization reagent.
  • PEI linear and/or branched polyethyleneimines
  • PPA polyphosphoroamidates
  • PPA polyphosphoroamidates
  • block polycationic copolymers comprising PEG and PEI or PPA
  • graft polycationic copolymers comprising PEG and PEI or PPA
  • an anionic stabilization reagent an anionic stabilization reagent.
  • the present invention provides one or more polymeric nanoparticles comprising: (a) one or more block copolymers of polyethylene glycol-Z>- polyphophoramidate (PEG- ⁇ - ⁇ ), (b) at least one or more polyribonucleotide molecules, and (c) an anionic stabilization reagent.
  • present invention provides one or more polymeric
  • nanoparticles comprising: (a) one or more graft copolymers of polyethyleneimine-g-polyethylene glycol (PEI-g-PEG), (b) at least one or more polyribonucleotide molecules, and (c) an anionic stabilization reagent.
  • PEI-g-PEG polyethyleneimine-g-polyethylene glycol
  • the present invention provides one or more polymeric nanoparticles comprising: (a) one or more polycationic polymers, (b) at least one or more polyribonucleotide molecules, and (c) an anionic stabilization reagent, wherein the polyribonucleotide molecule is a single stranded RNA molecule, a double stranded RNA molecule, a micro-RNA (miRNA) molecule, a short-hairpin RNA (shRNA) molecule, or a siRNA molecule.
  • miRNA micro-RNA
  • shRNA short-hairpin RNA
  • the present invention provides a method for making one or more polymeric nanoparticles having (a) one or more polycationic polymers, (b) at least one or more polyribonucleotide molecules, and (c) an anionic stabilization reagent, said method comprising: 1 ) obtaining a solution comprising at least one or more polyribonucleotide molecules, 2) adding to the solution of (1), a sufficient quantity of a solution containing an anionic stabilization reagent, and mixing the resulting solution; and 3) adding to the solution of (2), a sufficient quantity of a solution containing one or more polycationic polymers, mixing the solution, and incubating the resulting solution for a sufficient time to allow a complexation reaction to occur and the nanoparticles to assemble.
  • the present invention provides a method for making one or more polymeric nanoparticles having (a) one or more polycationic polymers selected from the group consisting of linear or branched polycationic homopolymers, block polycationic copolymers and graft polycationic copolymers, (b) at least one or more polyribonucleotide molecules, and (c) an anionic stabilization reagent, said method comprising: 1 ) obtaining a solution comprising at least one or more polyribonucleotide molecules, 2) adding to the solution of ( 1 ), a sufficient quantity of a solution containing an anionic stabilization reagent, and mixing the resulting solution; and 3) adding to the solution of (2), a sufficient quantity of a solution containing one or more polycationic polymers, mixing the solution, and incubating the resulting solution for a sufficient time to allow a complexation reaction to occur and the nanoparticles to assemble.
  • a polycationic polymers selected from the group consisting of linear or branched poly
  • the present invention provides a method for making one or more polymeric nanoparticles having (a) one or more block copolymers of polyethylene glycol- 6-polyphophoramidate (PEG-6-PPA), (b) at least one polynucleotide molecule; and (c) an anionic stabilization reagent, said method comprising: 1 ) obtaining a solution comprising at least one or more polyribonucleotide molecules, 2) adding to the solution of ( 1 ), a sufficient quantity of a solution containing an anionic stabilization reagent, and mixing the resulting solution; and 3) adding to the solution of (2), a sufficient quantity of a solution containing PEG-6-PPA, mixing the solution, and incubating the resulting solution for a sufficient time to allow a complexation reaction to occur and the nanoparticles to assemble.
  • PEG-6-PPA polyethylene glycol- 6-polyphophoramidate
  • the present invention provides a method of modulating expression of a target gene in a cell, or population of cells, comprising administering to the cell or population of cells, one or more polymeric nanoparticles comprising: (a) one or more polycationic polymers, (b) at least one or more polyribonucleotide molecules, and (c) an anionic stabilization reagent, in an amount sufficient to modulate target gene expression with the cell or population of cells.
  • the cells are mammalian cells.
  • the present invention provides a method of modulating expression of a target gene in a cell or population of cells comprising administering to the cell, or population of cells, one or more polymeric nanoparticles having: (a) one or more polycationic polymers selected from the group consisting of linear or branched polycationic homopolymers, block polycationic copolymers and graft polycationic copolymers, (b) at least one or more polyribonucleotide molecules, and (c) an anionic stabilization reagent, in an amount sufficient to modulate target gene expression with the cell, or population of cells.
  • one or more polymeric nanoparticles having: (a) one or more polycationic polymers selected from the group consisting of linear or branched polycationic homopolymers, block polycationic copolymers and graft polycationic copolymers, (b) at least one or more polyribonucleotide molecules, and (c) an anionic stabilization reagent, in an amount sufficient to modulate target gene expression
  • the present invention provides a method of modulating expression of a target gene in a cell, or population of cells, comprising administering to the cell, or population of cells, one or more polymeric nanoparticles having: (a) one or more block copolymers of PEG-6-PPA, (b) at least one or more polyribonucleotide molecules, and (c) an anionic stabilization reagent, in an amount sufficient to modulate target gene expression with the cell or population of cells.
  • the present invention provides a method of modulating expression of a target gene in a cell, or population of cells, comprising administering to the cell, or population of cells, one or more polymeric nanoparticles having: (a) one or more block copolymers of PEI-g-PEG, (b) at least one or more polyribonucleotide molecules, and (c) an anionic stabilization reagent, in an amount sufficient to modulate target gene expression with the cell or population of cells.
  • the present invention provides a use of a composition of one or more polymeric nanoparticle comprising: (a) one or more polycationic polymers, (b) at least one or more polyribonucleotide molecules, and (c) an anionic stabilization reagent, in an effective amount, wherein the composition includes a pharmaceutically and physiologically acceptable carrier, to prepare a medicament, preferably for use as a medicament for treating a disease in a subject.
  • the present invention provides a use of a composition of one or more polymeric nanoparticle comprising: (a) one or more polycationic polymers selected from the group consisting of linear or branched polycationic homopolymers, block polycationic copolymers and graft polycationic copolymers, (b) at least one or more polyribonucleotide molecules, and (c) an anionic stabilization reagent, in an effective amount, wherein the composition includes a pharmaceutically and physiologically acceptable carrier, to prepare a medicament, preferably for use as a medicament for treating a disease in a subject.
  • the present invention provides a use of a composition of one or more polymeric nanoparticle comprising: (a) one or more polycationic polymers selected from the group consisting of linear or branched polycationic homopolymers, block polycationic copolymers and graft polycationic copolymers, (b) at least one or more polyribonucleotide molecules, and (c) an anionic stabilization reagent, in an effective amount, wherein the composition includes a pharmaceutically and physiologically acceptable carrier, to prepare a medicament, preferably for use as a medicament for treating a disease in a subject.
  • the present invention provides a use of a composition of one or more polymeric nanoparticle comprising: (a) one or more block copolymers of polyethylene glycol-6-polyphophoramidate (PEG-6-PPA), (b) at least one or more polyribonucleotide molecules, and (c) an anionic stabilization reagent, in an effective amount, wherein the composition includes a pharmaceutically and physiologically acceptable carrier, to prepare a medicament, preferably for use as a medicament for treating a disease in a subject.
  • PEG-6-PPA polyethylene glycol-6-polyphophoramidate
  • an anionic stabilization reagent in an effective amount
  • the composition includes a pharmaceutically and physiologically acceptable carrier, to prepare a medicament, preferably for use as a medicament for treating a disease in a subject.
  • the present invention provides a method of modulating expression of a target gene in a mammalian cell or population of cells comprising administering to the cell, or population of cells, one or more polymeric nanoparticles having: (a) one or more polycationic polymers selected from the group consisting of linear polycationic polymers, (b) at least one or more polyribonucleotide molecules, and (c) an anionic stabilization reagent, in an amount sufficient to modulate target gene expression with the cell, or population of cells and diagnose the role of the target gene in a clinical condition or disease.
  • FIG. 1 depicts a schematic illustrating the synthesis of the polycationic block copolymer PEG-&-PPA in accordance with an embodiment of the present invention.
  • FIG. 2 depicts a schematic illustrating the synthesis of the self-assembling polymeric PEG-6-PPA/siRNA/TPP ternary nanoparticles formed through ionic crosslinking with the anionic stabilization reagent, sodium triphosphate (TPP), in accordance with an embodiment of the present invention.
  • TPP sodium triphosphate
  • FIG. 3 depicts a graph showing the mean particle hydrodynamic diameter (z-average) (left ordinate) and polydispersity index (PDI) (right ordinate) measured by dynamic light scattering (DLS) analysis of nanoparticles of the present invention formed at various P'/N ratios at a N/P ratio of 4 before ( ⁇ , o) and after ( ⁇ , ⁇ ) incubation with 150 mM NaCl for 24 hours.
  • z-average mean particle hydrodynamic diameter
  • PDI polydispersity index
  • FIG. 4 depicts a graph showing the z-average (left ordinate) and PDI (right ordinate) measured by dynamic light scattering analysis of an embodiment of the nanoparticles of the present invention formed at various P'/N ratios at a N/P ratio of 8 before ( ⁇ , o) and after ( ⁇ , ⁇ ) incubation with 1 50 mM NaCl for 24 hours.
  • FIG. 7 A is a graph depicting gene silencing efficiency mediated by PEG- PPA/siRNA/TPP ternary nanoparticles of an embodiment of the present invention at various P'/N ratios for N/P ratios of 4 and 8 in HeLa and D407 cells in the absence of 10% serum.
  • FIG 7B is a graph depicting gene silencing efficiency mediated by PEG- PPA siRNA/TPP ternary nanoparticles of an embodiment of the present invention at various P'/N ratios for N/P ratios of 4 and 8 in HeLa and D407 cells in the presence of 10% serum.
  • Fig. 9 is a series of transmission electron micrographs of (a) branched PEI/siRNA nanoparticles, (b) hyaluronic acid (HA)-coated branched PEI/siRNA nanoparticles, showing severe aggregation, (c) TPP stabilized branched PEI/siRNA nanoparticles, (d) HA-coated branched PEI/siRNA TPP nanoparticles, (e) TPP stabilized linear PEI/siRNA nanoparticles, and (f) HA-coated linear PEI/siRNA/TPP nanoparticles.
  • HA hyaluronic acid
  • Fig. 10 depicts a pair of bar graphs showing the gene knockdown efficiency (a), and cell viability (b) after treatment with PEI/siRNA nanoparticles of an embodiment of the present invention in HCE-2 cells (human corneal epithelial cell).
  • the present invention provides one or more self-assembling polymeric nanoparticles comprising a polynucleotide molecule, which has improved stability in salt and serum-containing solutions, and has the ability to protect incorporated polynucleotide molecules, such as siRNA, against enzymatic degradation in blood and other bodily fluids, while also having high efficiency in delivering the polynucleotide molecules to the target cytosol, and therefore can modulate the expression of a target gene of interest.
  • populations of cells and /or for use in diagnosing or treating a disease in a subject are also provided.
  • the present invention provides one or more polymeric nanoparticles comprising: (a) one or more polycationic homopolymers, (b) at least one or more
  • polycationic homopolymers means polymer chains of repeating subunits that have cationic residues.
  • Polycationic polymers are polymers that contain net positively charged atom(s) or associated group(s) of atoms covalently linked to the polymer molecules. This definition includes, but is not limited to phosphonium, sulfonium, and ammonium cations.
  • Other examples of cationic groups that can be covalently linked include, but are not limited to, amines (primary, secondary, tertiary, and aromatic) isocyanates,
  • polyacrylamides polyisobutylene, poly(N-vinylcarbazole), and polyquaternium polymers.
  • Polycationic homopolymers useful in accordance with the present invention include, for example, polymers such as linear or branched homopolymers, including, for example, linear and/or branched polyethyleneimines and derivatives thereof, and polyphosphoroamidates and derivatives thereof.
  • the present invention provides one or more polymeric nanoparticles comprising: (a) one or more polycationic polymers selected from the group consisting of linear or branched PEI, PPA, block polycationic copolymers comprising PEG and PEI or PPA and derivatives thereof, and graft polycationic copolymers comprising PEG and PEI or PPA and derivatives thereof, (b) at least one or more polyribonucleotide molecules, and (c) an anionic stabilization reagent.
  • polycationic block and graft copolymers and their derivatives can also be used in the nanoparticles, and include, for example, polyethylene glycol polymers.
  • block copolymers useful in the present invention include, PEG-i-PPA, and derivatives thereof, and examples of polycationic graft copolymers useful in the present invention include, PEI-g-PEG and derivatives thereof.
  • the present invention provides one or more polymeric nanoparticles comprising: (a) one or more
  • polycationic polymers (b) at least one or more polyribonucleotide molecules, and (c) an anionic stabilization reagent, wherein when there are two or more polycationic polymers.
  • the present invention provides one or more polymeric nanoparticles comprising: (a) one or more linear and or branched PEI polymers or derivatives thereof, (b) at least one or more polyribonucleotide molecules, and (c) an anionic stabilization reagent.
  • the present invention provides one or more polymeric nanoparticles comprising: (a) one or more linear and/or branched PPA polymers or derivatives thereof, (b) at least one or more polyribonucleotide molecules, and (c) an anionic stabilization reagent.
  • the polycationic polymers including linear and branched polymers, as well as the block and graft copolymers used in various embodiments of the present invention, are derivatives of polycationic polymers that include biocompatible polymers (that is, polymers that do not cause significant undesired physiological reactions), that can be either biodegradable or non-biodegradable polymers or blends or copolymers thereof.
  • biocompatible polymers that is, polymers that do not cause significant undesired physiological reactions
  • biocompatible polymers include, but are not limited to, polycationic biodegradable PPA.
  • the block copolymer of the present invention is the PEG- ⁇ - ⁇ copolymer. The synthesis of an embodiment of the PEG-6-PPA copolymer is shown in Fig. 1.
  • polycationic graft copolymers can be used.
  • the polycationic graft copolymer PEI-g-PEG is suitable for use with the nanoparticles of the present invention.
  • the present invention provides one or more polymeric nanoparticles comprising: (a) one or more polycationic graft copolymers of PEI-g-PEG, or derivatives thereof, (b) at least one or more polynucleotide molecules, and (c) an anionic stabilization reagent.
  • biodegradable refers to degradation in a biological system, for example enzymatic degradation or hydrolytic degradation.
  • anionic stabilization reagent means an inorganic or organic polyanionic molecule having a high charge density, and is capable of physically cross-linking the positively charged amino groups of the polycationic polymer chains of the nanoparticles, with the negatively charged polyanions of the anionic stabilization reagent.
  • charge density is defined herein as a ratio of a charge unit per unit of molecular weight.
  • a “charge unit” is defined as a molecular moiety having a positive or negative charge when in a solution at a pH range of between about 4 to about 12. Molecules having high charge density have a greater number of charge units per unit of molecular weight.
  • the molecule TPP has five negatively charged moieties in solution, and the anionic form ([ChPOPiO ⁇ OPCb] "5 ), and has a molecular weight of approximately 368 Daltons, thus TPP has a charge density of 1 anionic charge unit/74 Daltons.
  • the anionic stabilization reagent is sodium triphosphate (TPP).
  • TPP sodium triphosphate
  • Other possible examples of the anionic stabilization reagent known to those of skill in the art, including, for example, similar molecules (e.g., tetrasodium pyrophosphate and hexasodium metaphosphate), or other inorganic oligophosphates, in linear or cyclic chains ( « 1 - 4).
  • TPP sodium triphosphate
  • similar molecules e.g., tetrasodium pyrophosphate and hexasodium metaphosphate
  • the present invention provides one or more polymeric nanoparticles comprising: (a) one or more polycationic polymers, (b) at least one or more polynucleotide molecules, and (c) one or more anionic stabilization reagents.
  • the present invention provides a method for making one or more polymeric nanoparticles having (a) one or more polycationic polymers, (b) at least one or more polyribonucleotide molecules, and (c) an anionic stabilization reagent, said method comprising: 1 ) obtaining a solution comprising at least one or more polyribonucleotide molecules, 2) adding to the solution of ( 1 ), a sufficient quantity of a solution containing an anionic stabilization reagent, and mixing the resulting solution; and 3) adding to the solution of (2), a sufficient quantity of a solution containing one or more polycationic polymers of (a), mixing the solution, and incubating the resulting solution for a sufficient time to allow a complexation reaction to occur and the nanoparticles to assemble.
  • the present invention provides a method for making one or more polymeric nanoparticles having (a) one or more polycationic polymers selected from the group consisting of linear or branched polycationic homopolymers, block polycationic copolymers and graft polycationic copolymers, (b) at least one or more polyribonucleotide molecules, and (c) an anionic stabilization reagent, said method comprising: 1 ) obtaining a solution comprising at least one or more polyribonucleotide molecules, 2) adding to the solution of ( 1), a sufficient quantity of a solution containing an anionic stabilization reagent, and mixing the resulting solution; and 3) adding to the solution of (2), a sufficient quantity of a solution containing one or more polycationic polymers of (a), mixing the solution, and incubating the resulting solution for a sufficient time to allow a complexation reaction to occur and the nanoparticles to assemble.
  • a polycationic polymers selected from the group consisting of linear or branche
  • the present invention provides a method for making one or more polymeric nanoparticles having (a) one or more polycationic polymers selected from the group consisting of linear or branched polyethyleneimines (PEI), polyphosphoroamidates (PPA), block polycationic copolymers comprising PEG and PEI or PPA, and graft polycationic copolymers comprising PEG and PEI or PPA, and derivatives thereof, (b) at least one or more polyribonucleotide molecules, and (c) an anionic stabilization reagent, said method comprising: 1) obtaining a solution comprising at least one or more polyribonucleotide molecules, 2) adding to the solution of (1), a sufficient quantity of a solution containing an anionic stabilization reagent, and mixing the resulting solution; and 3) adding to the solution of (2), a sufficient quantity of a solution containing one or more polycationic polymers of (a), mixing the solution, and incubating the resulting solution for
  • PEI linear or branched
  • the present invention provides a method for making one or more polymeric nanoparticles having (a) one or more PEG- ⁇ - ⁇ polycationic block copolymers or derivatives thereof, (b) at least one or more polyribonucleotide molecules, and (c) an anionic stabilization reagent, said method comprising: 1 ) obtaining a solution comprising at least one or more polyribonucleotide molecules, 2) adding to the solution of (1 ), a sufficient quantity of a solution containing an anionic stabilization reagent, and mixing the resulting solution; and 3) adding to the solution of (2), a sufficient quantity of a solution containing one or more PEG-6-PPA polycationic block copolymers or derivatives thereof, mixing the solution, and incubating the resulting solution for a sufficient time to allow a complexation reaction to occur and the nanoparticles to assemble.
  • the present invention provides a method for making one or more polymeric nanoparticles having (a) one or more polycationic graft copolymers of PEI-g- PEG or derivatives thereof, (b) at least one or more polyribonucleotide molecules, and (c) an anionic stabilization reagent, said method comprising: 1) obtaining a solution comprising at least one or more polyribonucleotide molecules, 2) adding to the solution of (1), a sufficient quantity of a solution containing an anionic stabilization reagent, and mixing the resulting solution; and 3) adding to the solution of (2), a sufficient quantity of a solution containing one or more polycationic graft copolymers of PEI-g-PEG or derivatives thereof, mixing the solution, and incubating the resulting solution for a sufficient time to allow a complexation reaction to occur and the nanoparticles to assemble.
  • the term "sufficient time to allow a complexation reaction to occur and the nanoparticles to assemble,” means the time necessary for the one or more polyribonucleotide molecules, and an anionic stabilization reagent, to be mixed with the one or more polycationic polymers and have the self-assembly, or complexation to occur, and the nanoparticles to form.
  • the complexation reaction can occur within 5 minutes to an hour or more. Preferably, the complexation reaction occurs in less than an hour. More preferably, the complexation reaction occurs within about 10 minutes to about 30 minutes, and still more preferably, the complexation reaction occurs in about 15 minutes.
  • the time for complexation to occur can vary according to temperature, concentration of the reagents, and the type of polycationic polymers, anionic stabilization reagent and polyribonucleotide used.
  • the polymeric nanoparticles have a diameter between 30 nm and 300 nm, preferably between 40 nm and 150 nm, more preferably between 50 nm and 120 nm.
  • the diameter of the nanoparticles of the present invention can be measured by any means technically feasible that is known in the art. Preferred methods include photon correlation spectroscopy, doppler anemometry, light scattering measurement, and transmission electron microscopy.
  • the present invention provides that the one or more polymeric nanoparticles comprise at least one or more polynucleotide molecules which are between 10 bp and 10,000 bp in length, preferably between 20 bp and 50 bp in length, more preferably between 30 bp and 40 bp in length.
  • polynucleotide includes and/or is synonymous with “nucleic acid,” “oligonucleotide,” and “nucleic acid molecule,” and generally means a polymer of DNA or RNA, which can be single-stranded or double-stranded, synthesized or obtained (e.g., isolated and/or purified) from natural sources, which can contain natural, non-natural or altered nucleotides, and which can contain a natural, non-natural or altered intemucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide.
  • polyribonucleotide includes “ribonucleic acid,”
  • oligoribonucleotide and “ribonucleic acid molecule,” and generally means a polymer of RNA which can be single-stranded or double-stranded, synthesized or obtained (e.g., isolated and/or purified) from natural sources, which can contain natural, non-natural or altered nucleotides, and which can contain a natural, non-natural or altered intemucleotide linkage, such as a
  • nucleic acids may comprise one or more insertions, deletions, inversions, and/or substitutions.
  • the nucleic acids of the invention are recombinant.
  • the term “recombinant” refers to (i) molecules that are constructed outside living cells by joining natural or synthetic nucleic acid segments to nucleic acid molecules that can replicate in a living cell, or (ii) molecules that result from the replication of those described in (i) above.
  • the replication can be in vitro replication or in vivo replication.
  • the nucleic acids can be constructed based on chemical synthesis and/or enzymatic ligation reactions using procedures known in the art.
  • a nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed upon hybridization (e.g., phosphorothioate derivatives and acridine substituted nucleotides).
  • modified nucleotides that can be used to generate the nucleic acids include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5- iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl) uracil, 5- carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N 6 -isopentenyladenine, 1 -methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N 6 -substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl- 2-thiouracil, beta-D-man
  • the present invention provides one or more polymeric nanoparticles, wherein the polyribonucleotide molecule is selected from the group consisting of single stranded RNA, double stranded RNA, micro-RNA (miRNA), short-hairpin RNA
  • the present invention provides one or more polymeric nanoparticles comprising: (a) one or more
  • polycationic polymers (b) at least one or more polyribonucleotide molecules, and (c) an anionic stabilization reagent, wherein the polyribonucleotide molecule is a single stranded RNA molecule, a double stranded RNA molecule, a miRNA molecule, a shRNA molecule, or a siRNA molecule.
  • the present invention provides one or more polymeric nanoparticles comprising: (a) one or more polycationic polymers selected from the group consisting of linear polycationic homopolymers, block polycationic copolymers and graft polycationic copolymers, (b) at least one or more polyribonucleotide molecules, and (c) an anionic stabilization reagent, wherein the polyribonucleotide molecule is a single stranded RNA molecule, a double stranded RNA molecule, a miRNA molecule, shRNA molecule, or a siRNA molecule.
  • the present invention provides one or more polymeric nanoparticles comprising: (a) one or more polycationic polymers selected from the group consisting of linear and/or branched PEI, PPA, block polycationic copolymers comprising PEG and PEl or PPA, and graft polycationic copolymers comprising PEG and PEI or PPA, (b) at least one or more polyribonucleotide molecules, and (c) an anionic stabilization reagent, wherein the polyribonucleotide molecule is a single stranded RNA molecule, a double stranded RNA molecule, a miRNA molecule, a shRNA molecule, or a siRNA molecule.
  • the present invention provides one or more polymeric nanoparticles comprising: (a) PEG-&-PPA, and derivatives thereof, (b) at least one or more polyribonucleotide molecules, and (c) an anionic stabilization reagent, wherein the
  • polyribonucleotide molecule is a single stranded RNA molecule, a double stranded RNA molecule, a miRNA molecule, a shRNA molecule, or a siRNA molecule.
  • the present invention provides one or more polymeric nanoparticles comprising: (a) PEI-g-PEG, and derivatives thereof, (b) at least one or more polyribonucleotide molecules, and (c) an anionic stabilization reagent, wherein the
  • polyribonucleotide molecule is a single stranded RNA molecule, a double stranded RNA molecule, a miRNA molecule, a shRNA molecule, or a siRNA molecule.
  • the polyribonucleotides incorporated within the nanoparticles of the present invention can comprise any nucleotide sequence that encodes for a target gene of interest.
  • the present invention provides that the polynucleotide encodes for a
  • the telomere sequence of a target gene of interest in a cell or population of cells, either in vitro, or in vivo in a host.
  • the telomere sequence of a target gene of interest in a cell or population of cells, either in vitro, or in vivo in a host.
  • polynucleotide is an isolated or purified nucleic acid comprising a nucleotide sequence which is complementary to the nucleotide sequence of any target nucleotide sequence or a nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence of any of the nucleic acids described herein.
  • the present invention also relates to compounds, compositions, and methods useful for modulating the expression and activity of a target gene of interest, or expression and/or activity by RNAi using small nucleic acid molecules.
  • the instant invention features small nucleic acid molecules, or polyribonucleotides, and includes terms such as such as siRNA, siNA, dsRNA, miRNA, and shRNA molecules and methods used to modulate the expression of target genes of interest.
  • a polyribonucleotide of the invention can be unmodified or chemically modified.
  • a polyribonucleotide of the instant invention can be chemically synthesized, expressed from a vector or enzymatically synthesized.
  • the instant invention also features various chemically modified polyribonucleotides, including, for example, siRNA molecules capable of modulating repeat expansion gene expression or activity in cells by RNAi.
  • the use of chemically modified siRNA improves various properties of native siRNA molecules through increased resistance to nuclease degradation in vivo and/or through improved cellular uptake.
  • the polyribonucleotide molecule of the present invention comprises modified nucleotides while maintaining the ability to mediate RNAi.
  • the modified nucleotides can be used to improve in vitro or in vivo characteristics, such as stability, activity, and/or bioavailability.
  • the polyribonucleotide molecule is a siRNA molecule
  • the invention can comprise modified nucleotides as a percentage of the total number of nucleotides present in the siRNA molecule.
  • an siRNA molecule of the invention can generally comprise about 5% to about 100% modified nucleotides (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% modified nucleotides).
  • the actual percentage of modified nucleotides present in a given siRNA molecule will depend on the total number of nucleotides present in the siRNA. If the siRNA molecule is single-stranded, the percent modification can be based upon the total number of nucleotides present in the single-stranded siRNA molecules. Likewise, if the siRNA molecule is double- stranded, the percent modification can be based upon the total number of nucleotides present in the sense strand, antisense strand, or both the sense and antisense strands.
  • modulate means that the expression of the target gene, or level of RNA molecule or equivalent RNA molecules encoding one or more target proteins or protein subunits, or activity of one or more proteins or protein subunits is up regulated or down regulated, such that expression, level, or activity is greater than or less than that observed in the absence of the modulator.
  • modulate can mean “inhibit,” but the use of the word “modulate” is not limited to this definition.
  • inhibitor means that the expression of the target gene, or level of RNA molecules or equivalent RNA molecules encoding one or more target proteins or protein subunits, or activity of one or more target proteins or protein subunits, is reduced below that observed in the absence of the polyribonucleotide molecules (e.g., siRNA) of the invention.
  • inhibition, down-regulation or reduction with a siRNA molecule is below that level observed in the presence of an inactive or attenuated molecule.
  • inhibition, down- regulation, or reduction with siRNA molecules is below that level observed in the presence of, for example, a siRNA molecule with scrambled sequence or with mismatches.
  • inhibition, down-regulation, or reduction of target gene expression with a nucleic acid molecule of the instant invention is greater in the presence of the nucleic acid molecule than in its absence.
  • the amount of time of exposure of the nanoparticles to the host cells, population of cells or subject should be sufficiently long to effect gene "knockdown" or modulation of the expression of the target gene in the host cell, population of cells or in the subject.
  • the time for the desired effect varies with dosage, target, age and other factors known to those of skill in the art.
  • the time of exposure of the nanoparticles to the host cells, population of cells or subject should range from about 1 hour to about 120 hours, preferably from about 1 hour to about 48 hours, more preferably from about 1 hour to about 24 hours.
  • RNA nucleic acid that encodes a RNA
  • a gene or target gene can also encode a functional RNA (fRNA) or non-coding RNA (ncRNA), such as small temporal RNA (stRNA), miRNA, small nuclear RNA (snRNA), siRNA, small nucleolar RNA (snRNA), ribosomal RNA (rRNA), transfer RNA (tRNA) and precursor RNAs thereof.
  • fRNA small temporal RNA
  • miRNA small nuclear RNA
  • snRNA small nuclear RNA
  • siRNA small nucleolar RNA
  • rRNA ribosomal RNA
  • tRNA transfer RNA
  • Non-coding RNAs can serve as target nucleic acid molecules for siRNA mediated RNA interference in modulating the activity of fRNA or ncRNA involved in functional or regulatory cellular processes. Aberrant fRNA or ncRNA activity leading to disease can therefore be modulated by polyribonucleotide molecules of the invention.
  • Polyribonucleotide molecules targeting fRNA and ncRNA can also be used to manipulate or alter the genotype or phenotype of an organism or cell, by intervening in cellular processes such as genetic imprinting, transcription, translation, or nucleic acid processing (e.g., transamination, methylation etc.).
  • the target gene can be a gene derived from a cell, an endogenous gene, a transgene, or exogenous genes such as genes of a pathogen, for example a virus, which is present in the cell after infection thereof.
  • the term "complementarity" or “complementary” means that a nucleic acid can form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson- Crick or other non-traditional types.
  • the binding free energy for a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., RNAi activity. Determination of binding free energies for nucleic acid molecules is well known in the art (see, e.g., Turner et al., 1987, CSH Symp. Quant ' Biol. L1I pp.
  • a percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, or 10 nucleotides out of a total of 10 nucleotides in the first oligonucleotide being based paired to a second nucleic acid sequence having 10 nucleotides represents 50%, 60%, 70%, 80%, 90%, and 100% complementary respectively).
  • RNA means a molecule comprising at least one ribonucleotide residue.
  • ribonucleotide is meant a nucleotide with a hydroxyl group at the 2' position of a ⁇ -D-ribo-furanose moiety.
  • RNA ribonucleotides
  • polyribonucleotide also include double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of the siRNA, or internally, for example, at one or more nucleotides of the RNA.
  • Nucleotides in the RNA molecules of the instant invention can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.
  • the term "one or more polyribonucleotide molecules” means that the nanoparticles of the present invention can comprise more than one polyribonucleotide molecule.
  • the more than one polyribonucleotide molecules can include molecules having different nucleotide sequences directed to more than one mRNA nucleotide sequences.
  • the nanoparticles of the present invention can comprise polyribonucleotide molecules having, two, three or four distinct nucleotide sequences specific for different target genes or different sequences of the same target gene.
  • the present invention provides one or more polymeric nanoparticles wherein the polyribonucleotide molecule is a double stranded RNA molecule or siRNA.
  • the length of the siRNA molecule can be any length greater than about 10 bp, which is capable of binding its complementary sequence on the mRNA of the target gene of interest in the cytosol of a cell or population of cells.
  • the length of the siRNA can be about 20 to about 50 bp, including, for example, 20 bp, 25 bp, 30 bp, 35 bp, 40 bp, 45 bp, up to and including 50 bp.
  • any of the nanoparticle embodiments of the present invention described above can also encompass a pharmaceutical composition comprising the polymeric nanoparticles and a pharmaceutically acceptable carrier.
  • the present invention provides a pharmaceutical composition comprising one or more polymeric nanoparticles and a pharmaceutically acceptable carrier, wherein the nanoparticles comprise: (a) one or more polycationic homopolymers, (b) at least one or more polynucleotide molecules, and (c) an anionic stabilization reagent.
  • the present invention provides a pharmaceutical composition comprising one or more polymeric nanoparticles and a pharmaceutically acceptable carrier, wherein the nanoparticles comprise: (a) one or more polycationic polymers selected from the group consisting of linear and/or branched polyethyleneimines (PEI), polyphosphoroamidates (PPA), block polycationic copolymers comprising PEG and PEI or PPA, and graft polycationic copolymers comprising PEG and PEI or PPA, (b) at least one or more polynucleotide molecules, and (c) an anionic stabilization reagent.
  • PEI linear and/or branched polyethyleneimines
  • PPA polyphosphoroamidates
  • PPA polyphosphoroamidates
  • block polycationic copolymers comprising PEG and PEI or PPA
  • graft polycationic copolymers comprising PEG and PEI or PPA
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising one or more polymeric nanoparticles and a pharmaceutically acceptable carrier, wherein the nanoparticles comprise: (a) one or more polycationic block copolymers of PEG-6- PPA or derivatives thereof, (b) at least one or more polynucleotide molecules, and (c) an anionic stabilization reagent.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising one or more polymeric nanoparticles and a pharmaceutically acceptable carrier, wherein the nanoparticles comprise: (a) one or more polycationic graft copolymers of (PEI-g-PEG) or derivatives thereof, (b) at least one or more polynucleotide molecules, and (c) an anionic stabilization reagent.
  • the carrier can be any of those conventionally used, and is limited only by physico-chemical considerations, such as solubility and lack of reactivity with the active compound(s), and by the route of administration.
  • the carriers described herein for example, vehicles, adjuvants, excipients, and diluents, are well- known to those skilled in the art and are readily available to the public. It is preferred that the carrier be one which is chemically inert to the active agent(s), and one which has little or no detrimental side effects or toxicity under the conditions of use.
  • the carriers include soluble carriers such as known buffers which can be physiologically acceptable (e.g., phosphate buffer) as well as solid compositions such as solid-state carriers or latex beads.
  • the carriers or diluents used herein may be solid carriers or diluents for solid formulations, liquid carriers or diluents for liquid formulations, or mixtures thereof.
  • Solid carriers or diluents include, but are not limited to, gums, starches (e.g., corn starch, pregelatinized starch), sugars (e.g., lactose, mannitol, sucrose, dextrose), cellulosic materials (e.g., microcrystalline cellulose), acrylates (e.g., poly methylacry late), calcium carbonate, magnesium oxide, talc, or mixtures thereof.
  • pharmaceutically acceptable carriers may be, for example, aqueous or non-aqueous solutions, or suspensions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include, for example, water, alcoholic/aqueous solutions, cyclodextrins, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles for subcutaneous, intravenous, intraarterial, or intramuscular injection
  • parenteral vehicles include, for example, sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils.
  • Formulations suitable for parenteral administration include, for example, aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • Intravenous vehicles include, for example, fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like.
  • sterile liquids such as water and oils, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants.
  • water, saline, aqueous dextrose and related sugar solutions, and glycols such as propylene glycols or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.
  • the choice of carrier will be determined, in part, by the particular nanoparticle containing composition, as well as by the particular method used to administer the composition. Accordingly, there are a variety of suitable formulations of the pharmaceutical composition of the invention.
  • the following formulations for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal and interperitoneal administration are exemplary, and are in no way limiting. More than one route can be used to administer the compositions of the present invention, and in certain instances, a particular route can provide a more immediate and more effective response than another route.
  • injectable formulations are in accordance with the invention.
  • the requirements for effective pharmaceutical carriers for injectable compositions are well-known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Trissel, 1 5th ed., pages 622-630 (2009)).
  • the present invention provides one or more polymeric nanoparticles comprising: (a) one or more polycationic homopolymers, (b) at least one or more polynucleotide molecules, (c) an anionic stabilization reagent, and (d) one or more pharmaceutically active compounds.
  • the present invention provides one or more polymeric nanoparticles comprising: (a) one or more polycationic polymers selected from the group consisting of linear and/or branched PEI, PPA, block polycationic copolymers comprising PEG and PEI or PPA, and graft polycationic copolymers comprising PEG and PEI or PPA, (b) at least one or more polynucleotide molecules, (c) an anionic stabilization reagent, and (d) one or more pharmaceutically active compounds.
  • the present invention provides one or more polymeric nanoparticles comprising: (a) one or more polycationic block copolymers of PEG-6-PPA or derivatives thereof, (b) at least one or more polynucleotide molecules, (c) an anionic stabilization reagent, and (d) one or more pharmaceutically active compounds.
  • present invention provides one or more polymeric nanoparticles comprising: (a) one or more polycationic graft copolymers of PEl-g-PEG or derivatives thereof, (b) at least one or more polynucleotide molecules, (c) an anionic stabilization reagent, and (d) one or more pharmaceutically active compounds.
  • pharmaceutically active compound or “therapeutically active compound” means a compound useful for the treatment or modulation of a disease or condition in a subject suffering therefrom.
  • pharmaceutically active compounds can include any drugs known in the art for treatment of disease indications.
  • a particular example of a pharmaceutically active compound is a chemotherapeutic agent.
  • chemotherapeutic agent generally includes pharmaceutically or therapeutically active compounds that work by interfering with DNA synthesis or function in cancer cells. Based on their chemical action at a cellular level, chemotherapeutic agents can be classified as cell-cycle specific agents (effective during certain phases of cell cycle) and cell-cycle nonspecific agents (effective during all phases of cell cycle). Without being limited to any particular example, examples of chemotherapeutic agents can include alkylating agents, angiogenesis inhibitors, aromatase inhibitors,
  • antimetabolites anthracyclines, antitumor antibiotics, monoclonal antibodies, platinums, topoisomerase inhibitors, and plant alkaloids.
  • the amount or dose of the nanoparticles of the present invention that is administered should be sufficient to effectively target the cell, or population of cells in vivo, such that the modulation of the expression of the target gene of interest can be detected, in the subject over a reasonable time frame.
  • the dose will be determined by the efficacy of the particular nanoparticle formulation and the location of the target population of cells in the subject, as well as the body weight of the subject to be treated.
  • the dose of the nanoparticles of the present invention also will be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular nanoparticle. Typically, an attending physician will decide the dosage of the nanoparticle with which to treat each individual subject, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, compound to be administered, route of administration, and the severity of the condition being treated.
  • the dose of the nanoparticles of the present invention can be about 0.001 to about 1000 mg/kg body weight of the subject being treated, from about 0.01 to about 100 mg kg body weight, from about 0.1 mg/kg to about 10 mg/kg, and from about 0.5 mg to about 5 mg/kg body weight.
  • the dose of the nanoparticles of the present invention can be at a concentration from about 1 nM to about 10,000 nM, preferably from about 10 nM to about 5,000 nM, more preferably from about 100 nM to about 500 nM.
  • the N/P ratio of the nanoparticles of the present invention can vary in accordance with the amount the polycationic polymer added.
  • the amount of polycationic polymer added to the polynucleotide containing mixture can vary from none, to an amount that results in a ratio of 80, and any amount in between, for example, a N/P ratio of 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, to 20, can be achieved using the methods of the present invention.
  • the present invention provides one or more polymeric nanoparticles wherein the ratio of the number of primary amino groups of the polycationic polymer, for example, the PPA moiety to the number of phosphate groups in the polynucleotide molecule (N P ratio) is between 1 to 20, preferably between about 5 to about 15, more preferably between about 8 to 10.
  • the ratio of the number of phosphate groups of TPP to the number of primary amino groups in the polycationic polymer (PPA moiety) (P7N ratio) of the nanoparticles of the present invention can vary in accordance with the amount the anionic stabilization reagent added.
  • the present invention provides one or more polymeric nanoparticles wherein the ratio of the number of phosphate groups of TPP to the number of primary amino groups in the polycationic polymer, for example, the PPA moiety (P' N ratio) is between about 0.1 to about 1 .0, preferably between about 0.2 to about 0.8, more preferably about 0.5.
  • the present invention provides a method for making one or more polymeric nanoparticles, wherein the quantity of solution containing the polycationic polymer added is such that the ratio of the number of primary amino groups of the PPA moiety to the number of phosphate groups in the polynucleotide molecule (N P ratio) is between 1 to 20, preferably between about 5 to about 1 , more preferably between about 8 to 10.
  • the present invention provides a method for making one or more polymeric nanoparticles, wherein the quantity of solution containing the anionic stabilization reagent added is such that the ratio of the number of negatively charged groups of the anionic stabilization reagent to the number of positively charged groups in the polycationic polymer (P' N ratio) is between about 0 to about 1 .0, preferably between about 0.2 to about 0.8, more preferably about 0.5.
  • the polycationic polymer is PPA
  • the positively charged moieties are the amino groups.
  • the anionic stabilization reagent is TPP
  • the negatively charged groups are phosphate groups.
  • the present invention provides a method of modulating the expression of a target gene in a mammalian cell or population of cells comprising administering to the cell or population of cells, one or more polymeric nanoparticles, wherein the
  • polynucleotide molecule of the nanoparticles is a double stranded RNA molecule or siRNA, in an amount sufficient to modulating the target gene expression with the cell or population of cells.
  • the present invention provides a method of modulating the expression of a target gene in a host cell or population of cells comprising administering to the cell or population of cells, one or more polymeric nanoparticles, wherein the polynucleotide molecule of the nanoparticles is a double stranded RNA molecule or siRNA, in an amount sufficient to modulating the target gene expression with the cell or population of cells, wherein the target gene is a gene that when expression is increased, is associated with a disease.
  • the administering to a host cell or a population of cells, one or more polymeric nanoparticles of the present invention can be used to treat a disease in those cells. Examples of such diseases include, but are not limited to, neurological diseases, cardiovascular diseases, endocrine diseases, autoimmune diseases, gastrointestinal diseases, musculoskeletal diseases and cancer.
  • inventive methods can provide any amount of any level of treatment or prevention of cancer in a mammal.
  • the treatment or prevention provided by the inventive method can include treatment or prevention of one or more conditions or symptoms of the disease, e.g., cancer, being treated or prevented.
  • prevention can encompass delaying the onset of the disease, or a symptom or condition thereof.
  • the nanoparticles of the present invention can be designed to down regulate or inhibit target gene expression through RNAi targeting of a variety of RNA molecules.
  • the nanoparticles of the invention comprising siR A molecules are used to target various RNAs corresponding to a target gene.
  • RNAs include messenger RNA (mRNA), alternate RNA splice variants of target gene(s), post-transcriptionally modified RNA of target gene(s), pre-mRNA of target gene(s), and/or RNA templates.
  • the instant invention can be used to inhibit gene expression through the appropriate exons to specifically inhibit or to distinguish among the functions of gene family members.
  • a protein that contains an alternatively spliced transmembrane domain can be expressed in both membrane bound and secreted forms.
  • Use of the invention to target the exon containing the transmembrane domain can be used to determine the functional consequences of pharmaceutical targeting of membrane bound as opposed to the secreted form of the protein.
  • Non-limiting examples of applications of the invention relating to targeting these RNA molecules include therapeutic pharmaceutical applications, pharmaceutical discovery applications, molecular diagnostic and gene function applications, and gene mapping.
  • the invention further provides a host cell comprising any of the nanoparticles described herein.
  • the term "host cell” refers to any type of cell that can contain the inventive nanoparticles.
  • the host cell can be a eukaryotic cell, e.g., plant, animal, fungi, or algae.
  • the host cell can be a cultured cell or a primary cell, i.e., isolated directly from an organism, e.g., a human.
  • the host cell can be an adherent cell or a suspended cell, i.e., a cell that grows in suspension.
  • Suitable host cells are known in the art and include, for instance, HeLa cells (human epithelial cervical cancer cell line), D407 cells (human retinal pigmented epithelial cell line), Chinese hamster ovarian cells, monkey VERO cells, COS cells, HE 293 cells, and the like.
  • the host cell is preferably a mammalian cell. Most preferably, the host cell is a human cell.
  • suitable human host cells can include, but are not limited to, cells of the major organs of the body, including, for example, cells of the lung, including hepatocytes and hepatic stellate cells, cells of the breast, cells of the prostate, cells of the cornea, including corneal epithelial cells, cells of the lung, including lung epithelial cells, and cells of the brain, such as neurons. While the host cell can be of any cell type, can originate from any type of tissue, and can be of any combination of tissue, and can be of any combination of tissue.
  • the host cell preferably is a cancer cell.
  • the population of cells can be a heterogeneous population comprising the host cell comprising any of the nanoparticles described, in addition to at least one other cell, e.g., a host cell (e.g., a epithelial cell), which does not comprise any of the nanoparticles, or a cell other than a epithelial cell, e.g., a macrophage, a neutrophil, an erythrocyte, a hepatocyte, a hepatic stellate cell, an endothelial cell, an epithelial cell, a muscle cell, a brain cell, etc.
  • the population of cells can be a substantially homogeneous population, in which the population comprises mainly of host cells (e.g., consisting essentially of) comprising the nanoparticles.
  • the present invention provides a use of a composition comprising one or more polymeric nanoparticles comprising: (a) one or more polycationic polymers, (b) at least one or more polynucleotide molecules, and (c) an anionic stabilization reagent, in an effective amount, wherein the composition includes a pharmaceutically and physiologically acceptable carrier, to prepare a medicament, preferably for use as a medicament for treating a disease in a subject.
  • the present invention provides a use of a composition comprising one or more polymeric nanoparticles comprising: (a) one or more polycationic homopolymers, (b) at least one or more polynucleotide molecules, and (c) an anionic
  • composition includes a
  • a medicament preferably for use as a medicament for treating a disease in a subject.
  • the present invention provides a use of a composition comprising one or more polymeric nanoparticles comprising: (a) one or more polycationic polymers selected from the group consisting of linear and/or branched PEI, PPA, block polycationic copolymers comprising PEG and PEI or PPA, and graft polycationic copolymers comprising PEG and PEI or PPA, (b) at least one or more polynucleotide molecules, and (c) an anionic stabilization reagent, in an effective amount, wherein the composition includes a pharmaceutically and physiologically acceptable carrier, to prepare a medicament, preferably for use as a medicament for treating a disease in a subject.
  • a composition comprising one or more polymeric nanoparticles comprising: (a) one or more polycationic polymers selected from the group consisting of linear and/or branched PEI, PPA, block polycationic copolymers comprising PEG and PEI or PPA, and graft polycationic copolymers comprising P
  • the present invention provides a use of a composition comprising one or more polymeric nanoparticles comprising: (a) one or more block copolymers of PEG-6-PPA, (b) at least one or more polynucleotide molecules, and (c) an anionic
  • composition includes a
  • a medicament preferably for use as a medicament for treating a disease in a subject.
  • the present invention provides a use of a composition comprising one or more polymeric nanoparticles comprising: (a) one or more graft copolymers of PEI-g-PEG, (b) at least one or more polynucleotide molecules, and (c) an anionic stabilization reagent, in an effective amount, wherein the composition includes a pharmaceutically and physiologically acceptable carrier, to prepare a medicament, preferably for use as a medicament for treating a disease in a subject.
  • the medicament for treating a disease in a subject can encompass many different formulations known in the pharmaceutical arts, including, for example, intravenous and sustained release formulations.
  • the disease can include cancer.
  • Cancer can be any cancer, including any of acute lymphocytic cancer, acute myeloid leukemia, alveolar
  • rhabdomyosarcoma bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor.
  • the cancer is breast cancer and/or prostate cancer.
  • the nanoparticles of the present invention are useful in preparation of a medicament for treating cancers selected from the group consisting of melanoma, skin cancer, lung cancer, kidney cancer, stomach cancer, colon cancer, prostate cancer, breast cancer, ovarian cancer, and lymphoid cancer.
  • the present invention provides a composition comprising one or more polymeric nanoparticles comprising: (a) one or more polycationic polymers, (b) at least one or more polynucleotide molecules, and (c) an anionic stabilization reagent, in an amount effective for use in a medicament, and most preferably for use as a medicament for treating cancer, or inhibiting the growth of a tumor, or neoplasm in a subject, when administered to the subject in a therapeutically effective amount.
  • the present invention provides a composition comprising one or more polymeric nanoparticles comprising: (a) one or more polycationic polymers selected from the group consisting of linear and/or branched PEI, PPA, block polycationic copolymers comprising PEG and PEI or PPA, and graft polycationic copolymers comprising PEG and PEI or PPA, (b) at least one or more polynucleotide molecules, and (c) an anionic stabilization reagent, in an amount effective for use in a medicament, and most preferably for use as a medicament for treating cancer, or inhibiting the growth of a tumor, or neoplasm in a subject, when administered to the subject in a therapeutically effective amount.
  • the present invention provides a composition comprising one or more polymeric nanoparticles comprising: (a) one or more polycationic homopolymers, (b) at least one or more polynucleotide molecules, and (c) an anionic stabilization reagent, in an amount effective for use in a medicament, and most preferably for use as a medicament for treating cancer, or inhibiting the growth of a tumor, or neoplasm in a subject, when administered to the subject in a therapeutically effective amount.
  • the present invention provides a composition comprising one or more polymeric nanoparticles comprising: (a) one or more polycationic homopolymers, (b) at least one or more polynucleotide molecules, and (c) an anionic stabilization reagent, in an amount effective for use in a medicament, and most preferably for use as a medicament for treating cancer, or inhibiting the growth of a tumor, or neoplasm in a subject, when administered to the subject in a therapeutically effective amount.
  • the present invention provides a composition comprising one or more polymeric nanoparticles comprising: (a) one or more polycationic block copolymers of PEG-6-PPA or derivatives thereof, (b) at least one or more polynucleotide molecules, (c) an anionic stabilization reagent, and (d) one or more pharmaceutically active compounds, in an amount effective for use in a medicament, and most preferably for use as a medicament for treating cancer, or inhibiting the growth of a tumor, or neoplasm in a subject, when administered to the subject in a therapeutically effective amount.
  • the present invention provides a composition comprising one or more polymeric nanoparticles comprising: (a) one or more polycationic graft copolymers of polyethyleneimine-g-polyethylene glycol (PEI-g-PEG) or derivatives thereof, (b) at least one or more polynucleotide molecules, (c) an anionic stabilization reagent, and (d) one or more pharmaceutically active compounds, in an amount effective for use in a medicament, and most preferably for use as a medicament for treating cancer, or inhibiting the growth of a tumor, or neoplasm in a subject, when administered to the subject in a therapeutically effective amount.
  • PEI-g-PEG polyethyleneimine-g-polyethylene glycol
  • administering means that the one or more nanoparticles of the present invention are introduced into a sample having at least one cell, or population of cells, having a target gene of interest, and appropriate enzymes or reagents, in a test tube, flask, tissue culture, chip, array, plate, microplate, capillary, or the like, and incubated at a temperature and time sufficient to permit uptake of the at least one nanoparticles of the present invention into the cytosol, where it will bind to the mRNA of the target gene of interest and attenuate the expression of the target gene in the at least one cell or population of cells.
  • administering means that at least one or more nanoparticles of the present invention are introduced into a subject, preferably a subject receiving treatment for a disease, and the at least one or more nanoparticles are allowed to come in contact with the one or more disease related cells or population of cells having the target gene of interest in vivo.
  • treat includes diagnostic and preventative as well as disorder remitative treatment.
  • the term "subject” refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). It is more preferred that the mammals are from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). It is most preferred that the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). An especially preferred mammal is the human.
  • mammals of the order Rodentia such as mice and hamsters
  • mammals of the order Logomorpha such as rabbits. It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). It is
  • the nanoparticles of the present invention can be used in combination with one or more additional therapeutically active agents which are known to be capable of treating conditions or diseases discussed above.
  • the described nanoparticles of the present invention could be used in combination with one or more known therapeutically active agents, to treat a disease or condition.
  • Non-limiting examples of other therapeutically active agents that can be readily combined in a pharmaceutical composition with the nanoparticles of the present invention are enzymatic nucleic acid molecules, allosteric nucleic acid molecules, antisense, decoy, or aptamer nucleic acid molecules, antibodies such as monoclonal antibodies, small molecules, and other organic and/or inorganic compounds including metals, salts and ions.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising, in an effective amount, one or more polymeric nanoparticles, and pharmaceutically and physiologically acceptable carrier, wherein the nanoparticles comprise (a) one or more polycationic polymers, (b) at least one or more polynucleotide molecules, and (c) an anionic stabilization reagent.
  • the present invention provides a use of the pharmaceutical composition comprising, in an effective amount, one or more polymeric nanoparticles, and pharmaceutically and physiologically acceptable carrier, wherein the nanoparticles comprise (a) one or more polycationic polymers, (b) at least one or more polynucleotide molecules, and (c) an anionic stabilization reagent, to prepare a medicament, preferably for use as a medicament for treating a disease in a subject.
  • the nanoparticles of the present invention can be used in a variety of diagnostic applications, such as in the identification of molecular targets (e.g., RNA) in a variety of applications, for example, in clinical, industrial, environmental, agricultural and/or research settings.
  • diagnostic use of siRNA containing nanoparticles involves utilizing reconstituted RNAi systems, for example, using cellular lysates or partially purified cellular lysates.
  • siRNA in nanoparticles of the present invention can be used as diagnostic tools to examine genetic drift and mutations within diseased cells or to detect the presence of endogenous or exogenous, for example viral, RNA in a cell.
  • siRNA activity allows the detection of mutations in any region of the molecule, which alters the base-pairing and three-dimensional structure of the target RNA.
  • siRNA molecules in the nanoparticles of the present invention, one can map nucleotide changes, which are important to RNA structure and function in vitro, as well as in cells and tissues. Cleavage of target RNAs with siRNA molecules can be used to inhibit gene expression and define the role of specified gene products in the progression of disease or infection, or other clinical condition. In this manner, other genetic targets can be defined as important mediators of the disease.
  • the present invention provides a use of the composition comprising one or more polymeric nanoparticles comprising: (a) one or more polycationic polymers, (b at least one or more polynucleotide molecules, and (c) an anionic stabilization reagent, in an effective amount, for use in diagnosing a disease or condition in a subject.
  • siRNA containing nanoparticles of the present invention include detection of the presence of mRNAs associated with a disease, infection, or related condition. Such RNA is detected by determining the presence of a cleavage product after treatment with a siRNA using standard methodologies, for example, fluorescence resonance emission transfer (FRET).
  • FRET fluorescence resonance emission transfer
  • TPP Sodium triphosphate
  • PVSK poly(vinyl sulfate) potassium salt
  • WST- 1 was purchased from Roche (Penzberg, Germany).
  • UltraPureTM DNase RNase-Free distilled water, Dulbecco's Modified Eagle's Medium (DMEM), Opti-MEMTM and Lipofectamine2000TM were purchased from Invitrogen (Carlsbad, CA, USA).
  • DMEM Dulbecco's Modified Eagle's Medium
  • Opti-MEMTM Opti-MEMTM
  • Lipofectamine2000TM were purchased from Invitrogen (Carlsbad, CA, USA).
  • the Dual-luciferase reporter assay system and pGL3-control and pRL-CMV vectors were purchased from Promega (Madison, WI, USA).
  • siRNA was purchased from Ambion (Austin, TX, USA).
  • Formvar-coated carbon grid was purchased from Electron Microscopy Sciences (Hatfield, PA, USA).
  • the sequences of siRNA against Photinus pyralis Iuciferase were as follows: sense 5 '- CUUACGCUGAGUACUUCGAdTdT-3 ' (SEQ ID NO: 1), antisense 5'- UCG AAGU ACUCAGCGUAAGdTdT-3 ' (SEQ ID NO: 2).
  • the cyclic monomer 4-methyl-2- oxo-2-hydro- l ,3,2-dioxaphospholane was prepared as reported previously in J. Am. Chem. Soc, 1950, 72:5491-5497.
  • N',N 9 -bis(trifluoroacetyl) dipropyltriamine was synthesized following the procedure reported by O'Sullivan et al. at Tetrahedron Lett., 1995, 36:3451-34
  • PEG-A-PPA block copolymer The polycationic PEG-6-PPA block copolymer solutions were prepared as described in J. Control Release, 2007, 122(3): 297-304. Briefly, the synthesis of PEG-6-PPA is summarized in Fig. 1.
  • PEG-0 ⁇ + macroinitiator (1) was prepared by reacting about 1 .0 g of methoxy PEG with potassium granules (over stoichiometry) in 50 ml of anhydrous THF for 8 hours under refluxing. The concentration of PEG-0 ⁇ K + was determined by titration using 50 mM HC1.
  • the polymerization of 4-alkyl-2-oxo-2-hydro- 1 ,3,2- dioxaphospholane was initiated by adding PEG-0 ⁇ + solution into the reaction vessel at a molar ratio of 1 :500. The mixture was stirred at room temperature for about 48 hours. The precursor polymer (2) was obtained by precipitation into anhydrous toluene followed by vacuum drying. The precursor polymer (2) (3.0 g) was then dissolved in 20 ml of anhydrous DMF under argon. To this solution was added 27.2 g of N ⁇ N ⁇ bisitrifluoroacetyl) dipropyltriamine, followed by addition of 10 ml of anhydrous triethylamine and 10 ml of anhydrous CCU.
  • the mixture was stirred at 0 °C for 30 minutes, then at room temperature for 24 hours.
  • the reaction mixture was then precipitated into ether and dried under vacuum to yield polymer (3).
  • the resulting residue was suspended in 25% ammonia solution and stirred at 60 °C for 16 hours.
  • the solution was concentrated and dialyzed in dialysis tubing (MWCO 3500, Spectrapor, Spectrum Labs, CA) against distilled water for 2 days with frequent water change.
  • the PEG- ⁇ - ⁇ (4) was obtained after lyophilization (0.6 g, yield 12%).
  • PEG- ⁇ - PPA molecular weights: PEG, 12 kDa, PPA, 38 kDa
  • PEG- ⁇ - PPA molecular weights: PEG, 12 kDa, PPA, 38 kDa
  • TPP solution 10 mM Tris-HCl, pH 7.4
  • PEG-6-PPA solution 10 mM Tris-HCI, pH 7.4
  • the volume and concentration of siRNA solution were fixed: 10 of 4 ⁇ siRNA dissolved in 10 mM Tris-HCI buffer (pH 7.4), as shown in Table 1 .
  • siRNA- incorporated nanoparticles of an embodiment of the present invention were incubated at 37 °C with 50% final concentration of fetal bovine serum (FBS) for 1 hour, and 4 hours, respectively. Samples were then incubated in 10 ⁇ of 50 mM EDTA for 5 minutes, and 10 ⁇ of 10 mM PVSK was added to displace the siRNA from the nanoparticles. The released siRNA was analyzed by electrophoresis on a 20% polyacrylamide gel prepared in 7 M urea and TBE buffer (0.089 M Tris base, 0.089 M boric acid, and 2 mM sodium EDTA, pH 8.3).
  • Polyacrylamide- urea gel (20%) was used due to its high efficiency in separating small fragments of possibly degraded siRNA. Electrophoresis was then carried out with I X TBE buffer at a constant voltage of 100 V for 1 hour. The siRNA bands were visualized under a UV transilluminator after staining for 40 minutes with a 1 : 10,000 dilution of SYBR-Green II RNA gel stain (Molecular Probes) in RNase-free water.
  • HeLa cells human epithelial cervical cancer cell line
  • D407 cells human retinal pigment epithelial cell line
  • 720 ng/well pGL3-control plasmid encoding Photinus pyralis luciferase (P-Luc) and 80 ng/well pRL-CMV plasmid encoding Renilla reniformis luciferase (R-Luc) were co-transfected to the cells with Lipofectamine2000 (TM), according to the manufacturer's instructions, and cells were further incubated for an additional 4 hours.
  • TM Lipofectamine2000
  • the medium was replaced with fresh serum-free medium or medium with 10% FBS and
  • Cell viability was determined using WST- 1 according to the manufacture's protocol. Incubation conditions were identical to those used in the transfection protocol.
  • PEG-6-PPA/siRNA nanoparticles were prepared with the stabilization reagent, TPP, added to the siRNA solution.
  • PEG-6-PPA solutions were incubated with a mixture of siRNA and TPP at N P ratios of 4 and 8 and a variety of P7N ratios of 0 to 1 to form polyelectrolyte complexes.
  • P' N 0
  • the ⁇ -potential values of nanoparticles prepared at different P'/N ratios varied significantly and appeared to be highly dependent on P/N ratio.
  • the ⁇ -potential values decreased sharply from +15 to - 1 .5 mV when the P'/N ratio increased from 0.1 to 1.0.
  • the near electrostatic neutrality of the particle surface at P' N ratio of 0.5 to 1 .0, with a PEG corona, may be advantageous in preventing nanoparticle aggregation when applied in physiological media.
  • Nanoparticle embodiments prepared with lower P'/N ratios showed increased sizes after incubating in 0.15 M NaCI.
  • nanoparticles prepared at higher P'/N ratios only showed slightly increased particle sizes (average size of 1 10 and 100 nm for N/P of 4 and 8, respectively) and low PDIs (Figs. 3 and 4).
  • siRNA of the nanoparticles of the present invention have improved stability in serum containing medium
  • a sample of nanoparticles of the present invention were incubated with medium containing 50% FBS at 37 °C, followed by gel electrophoresis, in order to analyze the integrity of siRNA within the nanoparticles. It was determined that siRNA without nanoparticles, that was incubated with 50% FBS, was significantly degraded. In contrast, siRNA recovered from the nanoparticles of the present invention showed only trace amounts of degradation for all N/P and P' N ratios. The siRNA recovered from nanoparticles both with, and without TPP, were still more intact for 4 hours at all the P' N ratios (data not shown).
  • the stability of the nanoparticles was also analyzed by characterizing the release profile of incorporated siRNA using a polyanion-exchange reaction (data not shown).
  • anionic polymers including anionic proteins, sulfated polysaccharides, nuclear chromatin and messenger RNA (mRNA) as essential cellular components. Exchange reaction of polycations with these negatively-charged polymers take place in a biological environment. As a result, siRNA is released from nanoparticles through the intermolecular exchange and facilitates a series of subsequent RNAi processes for gene silencing.
  • nanoparticles should be sufficiently stable to resist decomplexation and release of siRNA before they reach the cytosol of target cells. Therefore, maintaining a balanced complex stability is important to successful transfection and subsequent knockdown of the desired target.
  • Knockdown efficiency i.e., the ability of the siRNA incorporated within the nanoparticles of the present invention to attenuate the expression of a target gene of interest
  • two different cell types HeLa, and D407, in the absence (Fig. 7A) and presence of serum (Fig. 7B).
  • HeLa and D407 cells were transiently transfected, respectively, with both reporter genes P-Luc and R-Luc, followed by treatment with the nanoparticles of the present invention, prepared at different P'/N ratios (0 to 1 .0) with siRNA against P-Luc.
  • the expression level of R-Luc was used as an internal reference for the initial transgene expression level.
  • the inhibition of P-Luc expression was evaluated by measuring the relative expression ratio of P-Luc/R-Luc at a concentration of 100 nM siRNA.
  • PEG-Z>-PPA/siRNA complexes without TPP failed to show distinct nanoparticles in DLS, although they did produce an average knockdown efficiency of 24% (P-Luc) and 58% (R- Luc ) in HeLa cells, as well as a knockdown efficiency of 40% (P-Luc) and 56% (R-Luc) in D407 cells, in serum-free medium, at N/P ratios of 4 and 8, respectively.
  • TPP-crossl inked nanoparticles of an embodiment of the present invention resulted in significantly higher knockdown efficiencies at both N/P ratios in both cell lines. As the P'/N ratio increases, the gene knockdown efficiency increased gradually.
  • the knockdown efficiency reached the plateau of about 60% at P'/N ratio of 0.5 in HeLa cells, and the highest knockdown efficiency of about 60% at P'/N ratio of 1.0 in D407 cells (Fig. 7A).
  • the knockdown efficiency was higher at N/P of 8, and reached about 75 % of knockdown efficiency in both types of cells.
  • the knockdown efficiency for N/P ratio of 4 was about 10- 12% (P-Luc), and 23-34% (R-Luc) at N/P ratio of 8 in both cell lines.
  • the knockdown efficiency of TPP-crosslinked nanoparticles of the present invention was maintained, or slightly enhanced, as compared to that obtained in serum-free medium transfection.
  • the gene knockdown efficiency maintained similar level in HeLa cells and moderately increased in D407 cells.
  • the knockdown efficiency reached the plateau of about 50% in Hela cells, and about a maximum of 70% in D407 cells.
  • Higher knockdown efficiency was obtained at N/P of 8 for both cell lines, with 75% to 80% knockdown efficiency obtained in HeLa cells and D407 cells, respectively, at P'/N ratio of 1.
  • the potential cytotoxicity of the nanoparticles of the present invention was assessed in both HeLa cells and D407 cells under the same transfection conditions.
  • the cell viability was determined by WST assay (Roche Applied Science, Indianapolis, IN )using water-soluble tetrazolium salt (Fig. 8). Nearly 100% of cell viability was observed in all transfection conditions in HeLa cells, and a cell viability of over 90% was observed in D407 cells, under identical conditions. There was no significant difference in cell viability observed between nanoparticles prepared with and without TPP crosslinking. Similar cytotoxicity results were obtained under serum-free transfection condition (data not shown). These results demonstrate that the addition of TPP into nanoparticle assemblies does not influence their cytotoxicity.
  • Nanoparticles prepared with TPP as the stabilizing agent were uniform spherical particles with an average size of 107 nm and 84 nm for linear PEl/siRNA TPP and branched PEI/siRNA/TPP, respectively (data not shown).
  • Linear PEI (17 kDa) and branched PEI (25 kDa) were used.
  • HCE-2 cells human corneal epithelial cell
  • KSF serum-free keratinocyte medium
  • 720 ng/well pGL3-control plasmid encoding P-Luc and 80 ng/well pRL-CMV plasmid encoding R-Luc were co-transfected to the cells with Lipofectamine2000TM according to the
  • the cells were further incubated for 4 hours.
  • the medium was replaced with fresh medium, and nanoparticles containing siRNA (100 nM) against P-Luc were applied to each well and incubated for 4 hours. The medium was then replaced with complete media, followed by incubation at 37°C with a 5 % of carbon dioxide atm.
  • cells were rinsed with PBS and subjected to a luciferase expression assay using the Dual-Luciferase Reporter Assay System. For each assay, P-Luc and R-Luc luminescence was measured using FLUOstar OPTIMA plate reader after the addition of appropriate substrates.
  • Cell viability was determined using WST-1 according to the manufacture's protocol. Incubation conditions were identical to those used in the transfection protocol.
  • HA-coating on the nanoparticle surface significantly increased the transfection and gene knockdown efficiency (Fig 10 (a)), and reduced the cytotoxicity of the nanoparticles (Fig 10 (b)).
  • Fig 10 (a) the transfection and gene knockdown efficiency
  • Fig 10 (b) the cytotoxicity of the nanoparticles

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Abstract

La présente invention concerne une ou plusieurs nanoparticules de polymère stabilisées comprenant : (a) des polymères polycationiques, (b) au moins une ou plusieurs molécules de polyribonucléotide, et (c) un réactif de stabilisation anionique. Les nanoparticules de polymère stabilisées présentent une uniformité élevée avec une petite taille, et présentent une stabilité augmentée vis-à-vis d'une force ionique physiologique et d'un milieu contenant du sérum. L'efficacité de transfection et de silençage génique des nanoparticules de polymère stabilisées est nettement améliorée par rapport aux nanoparticules qui ne contiennent pas le réactif de stabilisation dans du milieu contenant du sérum. La présente invention concerne en outre des procédés de fabrication des nanoparticules de polymère stabilisées, des compositions pharmaceutiques comprenant celles-ci, et des procédés pour leur utilisation.
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