WO2011109698A1 - Formulations et procédés d'administration ciblée à des cellules phagocytaires - Google Patents

Formulations et procédés d'administration ciblée à des cellules phagocytaires Download PDF

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WO2011109698A1
WO2011109698A1 PCT/US2011/027165 US2011027165W WO2011109698A1 WO 2011109698 A1 WO2011109698 A1 WO 2011109698A1 US 2011027165 W US2011027165 W US 2011027165W WO 2011109698 A1 WO2011109698 A1 WO 2011109698A1
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composition
polynucleotide
rna
modified
hydrophobic
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PCT/US2011/027165
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English (en)
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Karen G. Bulock
Anastasia Khvorova
Jennifer Lapierre
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Rxi Pharmaceuticals Corporation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • 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
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5036—Polysaccharides, e.g. gums, alginate; Cyclodextrin
    • 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
    • A61K9/5005—Wall or coating material
    • A61K9/5063—Compounds of unknown constitution, e.g. material from plants or animals
    • 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
    • A61K9/5005—Wall or coating material
    • A61K9/5063—Compounds of unknown constitution, e.g. material from plants or animals
    • A61K9/5068—Cell membranes or bacterial membranes enclosing drugs

Definitions

  • This invention pertains to formulations for nucleic acid delivery.
  • Particles containing glucan shells have been demonstrated to be effective for uptake by phagocytic cells.
  • any cell type that expresses Dectin 1 receptor can be targeted with this type of delivery vehicle.
  • Such particles have been used to deliver a variety of pay loads to these cell types including small molecules, proteins and nucleic acids.
  • nucleic acids cationic polymers like PEI (polyethylenimine) have been used to trap the pay load in the glucan shell.
  • PEI polyethylenimine
  • this technology has several major limitations including a high level of toxicity (up to 90-95 % targeted cell death), reduced efficacy of uptake and limited functionality. Very tight complexation in these particles may result in lack of compound release. The limited functionality of these particles is also due to endosomal entrapment of the complexed compounds. It would be of great benefit to develop non-toxic, functional delivery vehicles for nucleic acids.
  • nucleic acid delivery Described herein is a novel approach to nucleic acid delivery which enables efficient loading of nucleic acids into glucan-containing particles, generating a new class of delivery vehicles.
  • the nucleic acid molecule has increased hydrophobicity and is complexed with a glucan-containing particle and optionally a lipid or amphiphilic molecule.
  • Methods and compositions described herein offer significant advantages over previous approaches to delivery of nucleic acid molecules in glucan-containing particles.
  • the particles associated with the invention have significantly reduced toxicity, are better recognized by phagocytic cells, and have more efficient payload release and efficacy.
  • compositions and methods described herein have widespread applications for in vivo delivery of nucleic acids.
  • compositions including a hydrophobic modified polynucleotide complexed with a glucan-containing particle.
  • the composition can further include a lipid or amphiphilic molecule.
  • the polynucleotide is RNA.
  • the polynucleotide is double stranded RNA.
  • the RNA is an sd-rxRNA or an rxRNAori.
  • Compositions associated with the invention can also include a complexing agent.
  • the complexing agent comprises an amphiphilic molecule or a lipid.
  • the composition is a pharmaceutical composition and includes a pharmaceutically acceptable carrier. In some embodiments, the composition is sterile.
  • the hydrophobic portion of the hydrophobic modified polynucleotide can be covalently linked to the polynucleotide.
  • the hydrophobic portion of the hydrophobic modified polynucleotide is a sterol.
  • the sterol is a cholesterol, a cholesteryl or modified cholesteryl residue.
  • the hydrophobic portion of the hydrophobic modified polynucleotide is selected from the group consisting of bile acids, cholic acid or taurocholic acid, deoxycholate, oleyl litocholic acid, oleoyl cholenic acid, glycolipids, phospholipids, sphingolipids, isoprenoids, vitamins, saturated fatty acids, unsaturated fatty acids, fatty acid esters, triglycerides, pyrenes, porphyrines, Texaphyrine, adamantane, acridines, biotin, coumarin, fluorescein, rhodamine, Texas-Red, digoxygenin, dimethoxytrityl, t-butyldimethylsilyl, t-butyldiphenylsilyl, cyanine dyes (e.g. Cy3 or Cy5), Hoechst 33258 dye, psoralen, and ibuprof
  • the hydrophobic portion of the hydrophobic modified polynucleotide can also be non-covalently linked to the polynucleotide.
  • the hydrophobic portion of the hydrophobic modified polynucleotide is a polycationic molecule.
  • the polycationic molecule is selected from the group consisting of protamine, arginine rich peptides, and spermine.
  • compositions comprising a glucan-containing particle, an amphiphilic molecule or lipid, and a modified polynucleotide.
  • the amphiphilic molecule or lipid is an amphiphilic peptide.
  • the amphiphilic peptide is selected from the group consisting of: MW11308 (Polytran branched His/Lys peptide: KKK[KHHHKHHHKHHHKHHHKH]4-HHHHN (SEQ ID NO:l)); MW3781 (Linear peptide 1: H2N-
  • KETWWETWWTEWSQPGRKKRRQRRRPPQ-OH (SEQ ID NO:2)); MW4230: (Linear peptide 2: H2N- KGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQ-OH (SEQ ID NO:3) and Endo-Porter.
  • the modified polynucleotide is a modified RNA, such as a modified double stranded RNA.
  • the modified polynucleotide comprises a hydrophobic modification.
  • the modified polynucleotide comprises a 2'-0-methyl modification.
  • the modified double stranded RNA is an rxRNAori or an sd-rxRNA.
  • the composition is a pharmaceutical composition and includes a pharmaceutically acceptable carrier.
  • the composition is sterile.
  • composition has a diameter of 2-4 microns.
  • the polynucleotide can be modified. In some embodiments, the polynucleotide is at least 40% modified. In certain embodiments, the polynucleotide is an isolated double stranded nucleic acid molecule including a guide strand that is 16-28 nucleotides long and has complementarity to a target gene, wherein the 3' terminal 10 nucleotides of the guide strand include at least two phosphate modifications, and wherein the guide strand has a 5' phosphate modification and includes at least one 2' O-methyl modification or 2'-fluoro modification, and a passenger strand that is 8-28 nucleotides long and has complementarity to the guide strand, wherein the passenger strand is linked to the hydrophobic molecule, wherein the guide strand and the passenger strand form the double stranded nucleic acid molecule.
  • the polynucleotide is an isolated double stranded nucleic acid molecule including a guide strand and a passenger strand, wherein the guide strand is from 16-29 nucleotides long and is substantially complementary to a target gene, wherein the passenger strand is from 8-14 nucleotides long and has complementarity to the guide strand, and wherein the guide stand has at least two chemical modifications.
  • the polynucleotide is an isolated double stranded nucleic acid molecule comprising a guide strand and a passenger strand, wherein the guide strand is from 16-29 nucleotides long and is substantially complementary to a target gene, wherein the passenger strand is from 8-14 nucleotides long and has complementarity to the guide strand, and wherein the guide stand has a single stranded 3' region that is 5 nucleotides or longer.
  • the polynucleotide is an isolated double stranded nucleic acid molecule comprising a guide strand and a passenger strand, wherein the guide strand is from 16-29 nucleotides long and is substantially complementary to a target gene, wherein the passenger strand is from 8-29 nucleotides long and has complementarity to the guide strand, and wherein the double stranded nucleic acid molecule optionally has 3' overhangs.
  • the guide strand is from 24-29 nucleotides long and the passenger strand is from 24-29 nucleotides long.
  • the polynucleotide is an isolated single stranded nucleic acid molecule of 16-29 nucleotides in length and is substantially complementary to a target gene.
  • the polynucleotide is selected from the group consisting of an antisense ODN, an antagomir, an antiMirs, and a PNA.
  • the composition has a diameter of 2-4 microns. In certain embodiments, between 40-100% of the nucleotides of the polynucleotide are chemically modified nucleotides.
  • aspects of the invention involve methods for delivering a polynucleotide to a cell, including: contacting a cell with a composition described herein to deliver the polynucleotide to the cell.
  • the method can be performed in vitro or in vivo.
  • the polynucleotide has at least a region of sequence complementarity to a target gene and induces RNAi of mRNA of the target gene.
  • methods of inducing RNAi include: administering to a subject an effective amount for inducing RNAi of mRNA of a target gene, any of the compositions associated with the invention, wherein the polynucleotide has at least a region of sequence complementarity to the target gene.
  • the subject is a human.
  • the target gene is PPIB, MAP4K4 or SOD1.
  • methods of inducing RNAi include: administering to a subject an effective amount for inducing RNAi of mRNA of a target gene, any of the compositions associated with the invention, wherein the polynucleotide has at least a region of sequence complementarity to the target gene, wherein the step of administering is systemic, intravenous, intraperitoneal, intradermal, topical, intranasal, inhalation, oral, intramucosal or intraocular.
  • administration of a composition associated with the invention can be systemic, intravenous, intraperitoneal, intradermal, topical or intraocular.
  • FIG. 1 is a graph demonstrating expression of MAP4K4 in Peritoneal Exudate Cells
  • RNA duplex and target gene are indicated below each bar.
  • FIG. 2 is a graph demonstrating expression of PPIB in PECs following delivery of NNTs formulated in GeRPs using Formulations A, B and C.
  • the RNA duplex and target gene are indicated below each bar.
  • the GeRPs contain lipids within the shell (Glucan particle batch YGP SAF + L) while in FIG 2B, the GeRPs do not contain lipids within the shell (Glucan particle batch YGP SAF).
  • FIG. 3 is a graph demonstrating expression of MAP4K4 in PECs following delivery of NNTs formulated in GeRPs using Formulations A, B and C. The RNA duplex and target gene are indicated below each bar. The GeRPs were delivered at 15 GeRPs/cell.
  • FIG. 4 is a graph demonstrating expression of MAP4K4 in PECs following delivery of NNTs formulated in GeRPs using Formulations A, B and C. A comparison is
  • FIG. 5 is a graph demonstrating expression of PPIB in PECs following delivery of NNTs formulated in GeRPs using Formulations A, B and C. The RNA duplex and target gene are indicated below each bar. A comparison is demonstrated of delivery of 5, 10, 15 or 20 GeRPs/cell.
  • FIG. 6 demonstrates complex formation between sd-rxRNA nano and rxRNAori with peptides. Branched peptide and Linear Peptide 1 are demonstrated.
  • FIG. 7 demonstrates complex formation between sd-rxRNA nano and rxRNAori with peptides. Linear Peptide 2 and Endo-Porter are demonstrated.
  • FIG. 8 is a graph demonstrating expression of PPIB in PECs following delivery of RNA/peptide complexes formulated in GeRPs. The RNA duplex and target gene are indicated below each bar. A comparison is demonstrated of delivery of 5, 10, 15 or 20 GeRPs/cell.
  • FIG. 9 is a graph demonstrating expression of MAP4K4 in PECs following delivery of RNA/peptide complexes formulated in GeRPs.
  • the RNA duplex and target gene are indicated below each bar.
  • a comparison is demonstrated of delivery of 5, 10, 15 or 20 GeRPs/cell.
  • FIG. 10 is a graph demonstrating expression of MAP4K4 in PECs following delivery of RNA/peptide complexes formulated in GeRPs.
  • the RNA duplex and target gene are indicated below each bar.
  • a comparison is demonstrated of delivery of 5, 10, 15 or 20 GeRPs/cell.
  • FIG. 11 is a graph demonstrating expression of PPIB in PECs following delivery of
  • RNA/peptide complexes formulated in GeRPs The RNA duplex and target gene are indicated below each bar. A comparison is demonstrated of delivery of 5 or 10 GeRPs/cell.
  • FIG. 12 is a graph demonstrating expression of MAP4K4 in PECs following delivery of RNA/peptide complexes formulated in GeRPs. The RNA duplex and target gene are indicated below each bar. A comparison is demonstrated of delivery of 20, 30, 40, 50, 75 or 100 GeRPs/cell.
  • FIG. 13 is a graph demonstrating expression of PPIB in PECs following delivery of RNA/peptide complexes formulated in GeRPs. The RNA duplex and target gene are indicated below each bar. A comparison is demonstrated of delivery of 20, 30, 40, 50, 75 or 100 GeRPs/cell.
  • FIG. 14 is a graph demonstrating expression of PPIB in PECs following delivery of RNA/peptide complexes formulated in GeRPs. The RNA duplex and target gene are indicated below each bar. A comparison is demonstrated of delivery of 20, 30, 40, 50, 75 or 100 GeRPs/cell.
  • FIG. 15 is a graph demonstrating expression of MAP4K4 in PECs following delivery of RNA/peptide complexes formulated in GeRPs. The RNA duplex and target gene are indicated below each bar. A comparison is demonstrated of delivery of 20, 30, 40, 50, 75 or 100 GeRPs/cell.
  • FIG. 16 is a graph demonstrating expression of MAP4K4 in PECs following delivery of RNA/peptide complexes formulated in GeRPs. The RNA duplex and target gene are indicated below each bar. A comparison of conditions with and without serum is demonstrated.
  • FIG. 17 is a graph demonstrating expression of PPIB in PECs following delivery of RNA/peptide complexes formulated in GeRPs. The RNA duplex and target gene are indicated below each bar. A comparison of conditions with and without serum is demonstrated.
  • FIG. 18 is a graph demonstrating expression of PPIB in PECs following delivery of RNA/peptide complexes formulated in GeRPs. The RNA duplex and target gene are indicated below each bar. A comparison of conditions with and without serum is demonstrated.
  • FIG. 19 is a graph demonstrating expression of MAP4K4 in PECs following delivery of RNA/peptide complexes formulated in GeRPs. The RNA duplex and target gene are indicated below each bar. A comparison of conditions with and without serum is demonstrated.
  • the invention is based, at least in part, on the surprising discovery that complexation of hydrophobic modified oligonucleotides with glucan particles leads to highly effectively uptake by cells such as phagocytic cells.
  • Such particles can optionally further contain compounds such as amphiphilic molecules or lipids that are neutral or slightly charged.
  • the use of hydrophobic modified oligonucleotides may provide significant advantages for oligonucleotide delivery such as efficient loading, applicability of different entrapment (complexing) molecules than those used for the loading of traditional polynucleotides, improved endosomal escape after cellular internalization, and improved cellular recognition by cells such as by phagocytic cells. Additionally, and significantly, these compounds are non-toxic. With previously described PEI-based loaded GeRPs, significant toxicity was observed whereas the novel compounds have reduced toxicity. Thus, methods and compositions described herein represent a significant advance for in vivo oligonucleotide delivery.
  • compositions of the invention enable efficient complexing of oligonucleotides with glucan containing particles.
  • Oligonucleotide associated with the invention are modified in a manner such that the hydrophobicity of the molecule is increased (for example a hydrophobic molecule is attached (covalently or non-covalently) to a hydrophobic molecule on the oligonucleotide terminus or a non-terminal nucleotide, base, sugar, or backbone).
  • Any oligonucleotides may be used in the compositions of the invention,
  • the oligonucleotides may be RNA, DNA, single stranded, double stranded etc.
  • several sd-rxRNA compounds as well as non-sd-rxRNA compounds have been synthesized and formulated according to the invention.
  • glucan particles described herein are recognized and taken up by phagocytic cells by orders of magnitude better than previously described PEI-containing particles.
  • the presence of charge in the PEI-containing particles may disrupt membrane interaction, preventing uptake of such particles by cells.
  • the added hydrophobicity of the particles described herein may assist in their recognition and uptake at the cell surface.
  • the particles described herein show significantly improved escape and efficacy after cellular uptake. Without wishing to be bound by any theory, this may be due at least in part to the increased hydrophobicity of the RNA molecules allowing them to promote endosomal escape.
  • Particles described herein have dynamic loading components, meaning that they have improved ability to bind and release RNA during formulation and delivery, relative to particles described previously, such as PEI-containing particles.
  • PEI-containing particles PEI is bound tightly and densely to the RNA molecule, preventing such complexes from efficiently releasing RNA in the cell.
  • the RNA molecule, or the RNA molecule complexed with one or more lipids and/or peptides forms more reversible complexes resulting in improved release of RNA in the cell.
  • compositions described herein are their lack of toxicity.
  • significant toxicity has been observed at a 5: 1 ratio of GeRPs per cell.
  • molecules described herein no toxicity is observed even at a 50: 1 ratio of GeRPs per cell.
  • toxicity of previously described particles may be due, at least in part, to the presence of molecules such as PEI.
  • compositions of the invention include a hydrophobic modified polynucleotide complexed with a glucan-containing particle and optionally a lipid or amphiphilic molecule.
  • a "hydrophobic modified polynucleotide” as used herein is a polynucleotide (described below) that has at least one modification that renders the polynucleotide more hydrophobic than the polynucleotide was prior to modification. The modification may be achieved by attaching (covalently or non-covalently) a hydrophobic molecule to the polynucleotide. In some instances the hydrophobic molecule is or includes a lipophilic group.
  • lipophilic group means a group that has a higher affinity for lipids than its affinity for water.
  • lipophilic groups include, but are not limited to, cholesterol, a cholesteryl or modified cholesteryl residue, adamantine, dihydrotesterone, long chain alkyl, long chain alkenyl, long chain alkynyl, olely-lithocholic, cholenic, oleoyl-cholenic, palmityl, heptadecyl, myrisityl, bile acids, cholic acid or taurocholic acid, deoxycholate, oleyl litocholic acid, oleoyl cholenic acid, glycolipids, phospholipids, sphingolipids, isoprenoids, such as steroids, vitamins, such as vitamin E, fatty acids either saturated or unsaturated, fatty acid esters, such as triglycerides, pyrenes, porphyrines, Texaphyrine, a
  • the cholesterol moiety may be reduced (e.g. as in cholestan) or may be substituted (e.g. by halogen).
  • a combination of different lipophilic groups in one molecule is also possible.
  • the hydrophobic molecule may be attached at various positions of the polynucleotide.
  • the hydrophobic molecule may be linked to the terminal residue of the polynucleotide such as the 3' of 5 '-end of the polynucleotide.
  • it may be linked to an internal nucleotide or a nucleotide on a branch of the polynucleotide.
  • the hydrophobic molecule may be attached, for instance to a 2'-position of the nucleotide.
  • the hydrophobic molecule may also be linked to the heterocyclic base, the sugar or the backbone of a nucleotide of the polynucleotide.
  • the hydrophobic molecule may be connected to the polynucleotide by a linker moiety.
  • the linker moiety is a non-nucleotidic linker moiety.
  • Non-nucleotidic linkers are e.g. abasic residues (dSpacer), oligoethyleneglycol, such as triethyleneglycol (spacer 9) or hexaethylenegylcol (spacer 18), or alkane-diol, such as butanediol.
  • the spacer units are preferably linked by phosphodiester or phosphorothioate bonds.
  • the linker units may appear just once in the molecule or may be incorporated several times, e.g. via phosphodiester, phosphorothioate, methylphosphonate, or amide linkages.
  • Typical conjugation protocols involve the synthesis of polynucleotides bearing an aminolinker at one or more positions of the sequence, however, a linker is not required.
  • the amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents.
  • the conjugation reaction may be performed either with the
  • polynucleotide still bound to a solid support or following cleavage of the polynucleotide in solution phase. Purification of the modified polynucleotide by HPLC typically results in a pure material.
  • the hydrophobic molecule is a sterol type conjugate, a
  • PhytoSterol conjugate cholesterol conjugate, sterol type conjugate with altered side chain length, fatty acid conjugate, any other hydrophobic group conjugate, and/or hydrophobic modifications of the internal nucleoside, which provide sufficient hydrophobicity to be incorporated into micelles.
  • sterols refers or steroid alcohols are a subgroup of steroids with a hydroxyl group at the 3 -position of the A-ring. They are amphipathic lipids synthesized from acetyl-coenzyme A via the HMG-CoA reductase pathway. The overall molecule is quite flat. The hydroxyl group on the A ring is polar. The rest of the aliphatic chain is non-polar. Usually sterols are considered to have an 8 carbon chain at position 17.
  • sterol type molecules refers or steroid alcohols, which are similar in structure to sterols. The main difference is the structure of the ring and number of carbons in a position 17 attached side chain.
  • PhytoSterols also called plant sterols
  • Plant sterols are a group of steroid alcohols, phytochemicals naturally occurring in plants.
  • PhytoSterols There are more then 200 different known PhytoSterols
  • Steprol side chain refers to a chemical composition of a side chain attached at the position 17 of sterol-type molecule.
  • sterols are limited to a 4 ring structure carrying a 8 carbon chain at position 17.
  • the sterol type molecules with side chain longer and shorter than conventional are described.
  • the side chain may branched or contain double back bones.
  • sterols useful in the invention include cholesterols, as well as unique sterols in which position 17 has attached side chain of 2-7 or longer then 9 carbons.
  • the length of the polycarbon tail is varied between 5 and 9 carbons.
  • conjugates may have significantly better in vivo efficacy, in particular delivery to liver.
  • polynucleotide may be bound to a protein, peptide or positively charged chemical that functions as the hydrophobic molecule.
  • proteins include protamine, dsRNA binding domain, and arginine rich peptides.
  • Exemplary positively charged chemicals include spermine, spermidine, cadaverine, and putrescine.
  • hydrophobic molecule conjugates may demonstrate even higher efficacy when combined with optimal chemical modification patterns of the polynucleotide, containing but not limited to hydrophobic modifications.
  • the sterol type molecule may be a naturally occurring PhytoSterols.
  • the polycarbon chain may be longer than 9 and may be linear, branched and/or contain double bonds.
  • Some PhytoSterol containing polynucleotide conjugates may be significantly more potent and active in delivery of polynucleotides to various tissues.
  • Some PhytoSterols may demonstrate tissue preference and thus be used as a way to delivery RNAi specifically to particular tissues.
  • one or more bases of the polynucleotide is modified to increase hydrophobicity. Any base in any position might be modified, as long as modification results in an increase of the partition coefficient of the base.
  • the preferred locations for modification chemistries are positions 4 and 5 of the pyrimidines.
  • base modifications include 5-position uridine and cytidine modifications such as phenyl, methyl, 4-pyridyl, 2-pyridyl, indolyl, and isobutyl, phenyl (C6H50H);
  • tryptophanyl C8H6N)CH2CH(NH2)CO
  • Isobutyl butyl, aminobenzyl
  • phenyl phenyl
  • naphthyl
  • Hydrophobic modified oligonucleotides associated with the invention are complexed with glucan containing particles to form "GeRPs" (glucan encapsulated RNA loaded particle).
  • GeRPs glucan encapsulated RNA loaded particle.
  • Such particles are described and incorporated by reference from WO 2006/007372, WO 2007/050643, and US Patent Publications 2005/0281781 and 2010/0040656.
  • a GeRP refers to a particle that contains one or more glucans as a structural element and that can complex with an RNA molecule.
  • Glucan particles can be derived from insoluble components of fungal cell walls such as yeast cell walls.
  • the yeast is Baker's yeast.
  • Yeast-derived glucan molecules can include one or more of 6-(l,3)-Glucan, 6-(l,6)-Glucan, mannan and chitin.
  • a glucan particle comprises a hollow yeast cell wall whereby the particle maintains a three dimensional structure resembling a cell, within which it can complex with or encapsulate a molecule such as an RNA molecule.
  • glucan particles can be prepared by extraction of insoluble components from cell walls, for example by extracting Baker's yeast (Fleischmann's) with 1M NaOH/pH 4.0 H20, followed by washing and drying. Methods of preparing yeast cell wall particles are discussed in, and incorporated by reference from U.S. Patents 4,810,646, 4,992,540, 5,082,936, 5,028,703, 5,032,401, 5,322,841, 5,401,727, 5,504,079, 5,607,677, 5,968,811, 6,242,594, 6,444,448, 6,476,003, US Patent Publications 2003/0216346,
  • Protocols for preparing glucan particles are also described in, and incorporated by reference from, the following references: Soto and Ostroff (2008), "Characterization of multilayered nanoparticles encapsulated in yeast cell wall particles for DNA delivery.” Bioconjug Chem 19(4): 840-8; Soto and Ostroff (2007), “Oral Macrophage Mediated Gene Delivery System,” Nanotech, Volume 2, Chapter 5 (“Drug Delivery”), pages 378-381; and Li et al. (2007), "Yeast glucan particles activate murine resident macrophages to secrete proinflammatory cytokines via MyD88-and Syk kinase-dependent pathways.” Clinical Immunology 124(2): 170-181.
  • Glucan containing particles such as yeast cell wall particles can also be obtained commercially.
  • Several non-limiting examples include: Nutricell MOS 55 from Biorigin (Sao Paolo, Brazil), SAF-Mannan (SAF Agri, Minneapolis, Minn.), Nutrex (Sensient
  • alkali-extracted particles such as those produced by Nutricepts (Nutricepts Inc., Burnsville, Minn.) and ASA Biotech, acid-extracted WGP particles from Biopolymer Engineering, and organic solvent-extracted particles such as AdjuvaxTMfrom Alpha-beta Technology, Inc. (Worcester, Mass.) and microparticulate glucan from Novogen (Stamford, Conn.).
  • Glucan particles such as yeast cell wall particles can have varying levels of purity depending on the method of production and/or extraction.
  • particles are alkali-extracted, acid-extracted or organic solvent-extracted to remove intracellular components and/or the outer mannoprotein layer of the cell wall.
  • Such protocols can produce particles that have a glucan (w/w) content in the range of 50% - 90%.
  • a particle of lower purity, meaning lower glucan w/w content may be preferred, while in other embodiments, a particle of higher purity, meaning higher glucan w/w content may be preferred.
  • Glucan particles such as yeast cell wall particles
  • the particles can have a natural lipid content.
  • the particles can contain 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% or more than 20% w/w lipid.
  • YGP SAF YGP SAF + L (containing natural lipids).
  • the presence of natural lipids may assist in complexation or capture of RNA molecules.
  • Glucan containing particles typically have a diameter of approximately 2-4 microns, although particles with a diameter of less than 2 microns or greater than 4 microns are also compatible with aspects of the invention.
  • RNA molecule(s) to be delivered are complexed or "trapped" within the shell of the glucan particle.
  • the shell or RNA component of the particle can be labeled for visualization, as described in, and incorporated by reference from, Soto and Ostroff (2008) Bioconjug Chem 19:840. Methods of loading GeRPs are discussed further below.
  • compositions comprising a glucan- containing particle, an amphiphilic molecule or lipid, and a modified polynucleotide.
  • the modified polynucleotide is hydrophobically modified, while in other embodiments, the modified polynucleotide is not hydrophobically modified.
  • Associated methods involve the use of amphiphilic molecules, such as amphiphilic peptides, for loading polynucleotides into glucan-containing particles.
  • an "amphiphilic molecule” also called an “amphipathic molecule” refers to a molecule that possesses both hydrophilic and lipophilic properties.
  • amphiphilic molecules include phospholipids, cholesterol, glycolipids, fatty acids, bile acids and saponin.
  • the amphiphilic molecule can be a peptide which can be linear or branched. As demonstrated in the Examples, several non-limiting examples of peptides that are functional in complexing with oligonucleotides include MW11308:
  • peptides are complexed with RNA molecules, such as sd-rxRNAnano or rxRNAori RNA in RNA:peptide w/w ratios such as 1: 1, 1 :2.5, 1 :5, 1:7.5, 1 : 10.
  • hydrophobic modified oligonucleotides are combined with one or more lipids that can be charged or neutral.
  • Liposome based formulations are widely used for oligonucleotide delivery. Most commercially available lipid or liposome formulations contain at least one positively charged lipid (cationic lipids).
  • liposome formulations containing cationic lipids are characterized by a high level of toxicity.
  • In vivo limited therapeutic indexes have revealed that liposome formulations containing positive charged lipids are associated with toxicity (i.e. elevation in liver enzymes) at concentrations only slightly higher than concentration required to achieve RNA silencing.
  • Aspects of the invention include hydrophobic modified polynucleotides encompassed within neutral nanotransporters. Further description of neutral nanotransporters is incorporated by reference from PCT Publication No. WO2010/033248 (Application No. PCT/US2009/005251), filed on September 22, 2009, and entitled "Neutral Nanotransporters.”
  • Such particles enable quantitative oligonucleotide incorporation into non- charged lipid mixtures.
  • nanotransporter compositions is an important feature.
  • oligonucleotides can effectively be incorporated into a lipid mixture that is free of cationic lipids and such a composition can effectively deliver a therapeutic oligonucleotide to a cell in a manner that it is functional.
  • a high level of activity was observed when the fatty mixture was composed of a phosphatidylcholine base fatty acid and a sterol such as a cholesterol.
  • one preferred formulation of neutral fatty mixture is composed of at least 20% of DOPC or DSPC and at least 20% of sterol such as cholesterol. Even as low as 1:5 lipid to oligonucleotide ratio was shown to be sufficient to get complete encapsulation of the oligonucleotide in a non charged formulation.
  • the hydrophobic modified polynucleotide is mixed with a neutral fatty mixture to form a micelle.
  • the neutral fatty acid mixture is a mixture of fats that has a net neutral or slightly net negative charge at or around physiological pH that can form a micelle with the hydrophobic modified polynucleotide.
  • the term "micelle" refers to a small nanoparticle formed by a mixture of non charged fatty acids and phospholipids.
  • the neutral fatty mixture may include cationic lipids as long as they are present in an amount that does not cause toxicity.
  • the neutral fatty mixture is free of cationic lipids.
  • a mixture that is free of cationic lipids is one that has less than 1% and preferably 0% of the total lipid being cationic lipid.
  • cationic lipid includes lipids and synthetic lipids having a net positive charge at or around physiological pH.
  • anionic lipid includes lipids and synthetic lipids having a net negative charge at or around physiological pH.
  • the neutral fats bind to the oligonucleotides of the invention by a strong but non- covalent attraction (e.g. , an electrostatic, van der Waals, pi-stacking, etc. interaction).
  • a strong but non- covalent attraction e.g. , an electrostatic, van der Waals, pi-stacking, etc. interaction.
  • the neutral fat mixture may include formulations selected from a class of naturally occurring or chemically synthesized or modified saturated and unsaturated fatty acid residues.
  • Fatty acids might exist in a form of triglycerides, diglycerides or individual fatty acids.
  • the use of well-validated mixtures of fatty acids and/or fat emulsions currently used in pharmacology for parenteral nutrition may be utilized.
  • the neutral fatty mixture is preferably a mixture of a choline based fatty acid and a sterol.
  • Choline based fatty acids include for instance, synthetic phosphocholine derivatives such as DDPC, DLPC, DMPC, DPPC, DSPC, DOPC, POPC, and DEPC.
  • DOPC (chemical registry number 4235-95-4) is dioleoylphosphatidylcholine (also known as dielaidoylphosphatidylcholine, dioleoyl-PC, dioleoylphosphocholine, dioleoyl-sn-glycero-3- phosphocholine, dioleylphosphatidylcholine).
  • DSPC (chemical registry number 816-94-4) is distearoylphosphatidylcholine (also known as l,2-Distearoyl-sn-Glycero-3-phosphocholine).
  • the sterol in the neutral fatty mixture may be for instance cholesterol.
  • the neutral fatty mixture may be made up completely of a choline based fatty acid and a sterol or it may optionally include a cargo molecule.
  • the neutral fatty mixture may have at least 20% or 25% fatty acid and 20% or 25% sterol.
  • the term "Fatty acids” relates to conventional description of fatty acid. They may exist as individual entities or in a form of two-and triglycerides.
  • fat emulsions refers to safe fat formulations given intravenously to subjects who are unable to get enough fat in their diet. It is an emulsion of soy bean oil (or other naturally occurring oils) and egg phospholipids. Fat emulsions are being used for formulation of some insoluble anesthetics.
  • fat emulsions might be part of commercially available preparations like Intralipid, Liposyn, Nutrilipid, modified commercial preparations, where they are enriched with particular fatty acids or fully de novo- formulated combinations of fatty acids and phospholipids.
  • the cells to be contacted with an oligonucleotide composition of the invention are contacted with a mixture comprising the oligonucleotide and a mixture comprising a lipid, e.g. , one of the lipids or lipid compositions described supra for between about 12 hours to about 24 hours.
  • the cells to be contacted with an oligonucleotide composition are contacted with a mixture comprising the oligonucleotide and a mixture comprising a lipid, e.g., one of the lipids or lipid compositions described supra for between about 1 and about five days.
  • the cells are contacted with a mixture comprising a lipid and the oligonucleotide for between about three days to as long as about 30 days.
  • a mixture comprising a lipid is left in contact with the cells for at least about five to about 20 days.
  • a mixture comprising a lipid is left in contact with the cells for at least about seven to about 15 days.
  • lipid or molecule can optionally be any other lipid or molecule.
  • a lipid or molecule is referred to herein as a cargo lipid or cargo molecule.
  • Cargo molecules include but are not limited to intralipid, small molecules, fusogenic peptides or lipids or other small molecules might be added to alter cellular uptake, endosomal release or tissue distribution properties. The ability to tolerate cargo molecules is important for modulation of properties of these particles, if such properties are desirable. For instance the presence of some tissue specific metabolites might drastically alter tissue distribution profiles. For example use of Intralipid type formulation enriched in shorter or longer fatty chains with various degrees of saturation affects tissue distribution profiles of these type of formulations (and their loads).
  • a cargo lipid useful according to the invention is a fusogenic lipid.
  • the zwiterionic lipid DOPE (chemical registry number 4004-5-1, 1,2-Dioleoyl-sn- Glycero-3-phosphoethanolamine) is a preferred cargo lipid.
  • Intralipid may be comprised of the following composition: 1 000 mL contain:
  • fat emulsion is Liposyn that contains 5% safflower oil, 5% soybean oil, up to 1.2% egg phosphatides added as an emulsifier and 2.5% glycerin in water for injection. It may also contain sodium hydroxide for pH adjustment. pH 8.0 (6.0 - 9.0). Liposyn has an osmolarity of 276 m Osmol/liter (actual).
  • Variation in the identity, amounts and ratios of cargo lipids affects the cellular uptake and tissue distribution characteristics of these compounds. For example, the length of lipid tails and level of saturability will affect differential uptake to liver, lung, fat and
  • cardiomyocytes Addition of special hydrophobic molecules like vitamins or different forms of sterols can favor distribution to special tissues which are involved in the metabolism of particular compounds. Complexes are formed at different oligonucleotide concentrations, with higher concentrations favoring more efficient complex formation.
  • the fat emulsion is based on a mixture of lipids. Such lipids may include natural compounds, chemically synthesized compounds, purified fatty acids or any other lipids.
  • the composition of fat emulsion is entirely artificial.
  • the fat emulsion is more then 70% linoleic acid.
  • the fat emulsion is at least 1% of cardiolipin.
  • Linoleic acid (LA) is an unsaturated omega-6 fatty acid. It is a colorless liquid made of a carboxylic acid with an 18-carbon chain and two cis double bonds.
  • the alteration of the composition of the fat emulsion is used as a way to alter tissue distribution of hydrophobic modified polynucleotides.
  • This methodology provides for the specific delivery of the polynucleotides to particular tissues.
  • the fat emulsions of the cargo molecule contain more then 70% of Linoleic acid (C18H3202) and/or cardiolipin are used for specifically delivering RNAi to heart muscle.
  • Fat emulsions like intralipid have been used before as a delivery formulation for some non-water soluble drugs (such as Propofol, re-formulated as Diprivan).
  • non-water soluble drugs such as Propofol, re-formulated as Diprivan.
  • molecules described herein contain features incorporated by reference from
  • PCT/US2009/005251 including (a) the concept of combining modified polynucleotides with the hydrophobic compound(s), so it can be incorporated in the fat micelles and (b) mixing it with the fat emulsions to provide a reversible carrier.
  • micelles After injection into a blood stream, micelles usually bind to serum proteins, including albumin, HDL, LDL and other. This binding is reversible and eventually the fat is absorbed by cells. The polynucleotide, incorporated as a part of the micelle will then be delivered closely to the surface of the cells. After that, cellular uptake may happen though variable mechanisms, including but not limited to sterol type delivery.
  • a hydrophobic modified polynucleotide can be loaded into a GeRP particle in a variety of ways.
  • the RNA particle is loaded into the GeRP without a lipid or amphiphilic molecule, while in other embodiments, it is loaded with a lipid or amphiphilic molecule such as an amphiphilic peptide.
  • an RNA molecule and a lipid can be loaded into a GeRP particle in a lipid-first or RNA-first order. In some embodiments, a higher trapping efficiency is achieved if loading is conducted in a lipid-first order.
  • a lipid mixture consisting of 50:50 DOPC holesterol in ethanol is loaded first, followed by loading of the RNA molecule.
  • the RNA can be added before the peptide or the peptide can be added before the RNA.
  • RNA can be loaded into a GeRP.
  • approximately 1.8 mg of RNA such as sd-rxRNA, is loaded into approximately 10 mg of GeRP.
  • 50:50 DOPC:cholesterol in ethanol is loaded first, followed by an sd-rxRNA, while in other embodiments, the sd-rxRNA is loaded first, followed by addition of 50:50 DOPC:cholesterol in ethanol.
  • approximately 1.8 mg of RNA, such as sd-rxRNA is loaded into approximately 10 mg of GeRP, followed by addition of a peptide, while in other embodiments, the peptide is added first, followed by addition of the sd-rxRNA.
  • the ratio of RNA:peptide or RNA:lipid can vary.
  • the ratio of RNA:peptide or RNA:lipid can be 1: 1, 1: 1.5, 1:2, 1 :2.5, 1:3, 1 :3.5, 1 :4, 1:4.5, 1:5, 1:5.5, 1:6, 1 :6.5, 1 :7, 1 :7.5, 1 :8, 1:8.5, 1 :9, 1 :9.5, 1: 10, or more than 1: 10.
  • the ratio of RNA:peptide or RNA:lipid is less than 1 : 1.
  • GeRPs associated with the invention can be transfected into cells such as PECs and tested for their ability to silence gene expression.
  • the number of GeRPs administered per cell can vary, both in vitro and in vivo. In some instances, a dose-response is observed whereby silencing of gene expression increases with an increased number of GeRPs within a cell. In some embodiments, approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125 or more than 125 GeRPs are administered to a cell.
  • polynucleotide includes any molecule that is an organic polymer molecule composed of nucleotide monomers covalently bonded in a chain.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • polynucleotide is used synonymously with oligonucleotide and nucleic acid.
  • Nucleotide refers to a ribonucleotide or a deoxyribonucleotide or modified form thereof, as well as an analog thereof.
  • Nucleotides include species that comprise purines, e.g., adenine, hypoxanthine, guanine, and their derivatives and analogs, as well as pyrimidines, e.g., cytosine, uracil, thymine, and their derivatives and analogs.
  • a "nucleotide” comprises a cytosine, uracil, thymine, adenine, or guanine moiety.
  • Polynucleotides include any such primers, probes, and/or oligomer fragments.
  • Polynucleotides include polydeoxyribonucleotides (containing 2-deoxy-D-ribose), to polyribonucleotides (containing D-ribose), and to any other type of polynucleotide which is an N glycoside of a purine or pyrimidine base, or modified purine or pyrimidine base.
  • polynucleotide includes any type of nucleic acid and/or oligonucleotide. These terms refer only to the primary structure of the molecule. Thus, these terms include double- and single-stranded DNA, as well as triple-, double-and single-stranded RNA.
  • RNA molecules include RNAi, siRNA, miRNA, sd-rxRNA and siRNA inhibitors, single stranded substrates for RISC assembly and the like. Some of these examples will be discussed in more detail below. Such discussion is exemplary only and is non-limiting.
  • deoxynucleotide refers to a nucleotide or polynucleotide lacking an OH group at the 2' or 3' position of a sugar moiety with appropriate bonding and/or 2', 3' terminal dideoxy, instead having a hydrogen bonded to the 2' and/or 3' carbon.
  • deoxyribonucleotide and “DNA” refer to a nucleotide or polynucleotide comprising at least one ribosyl moiety that has an H at its 2' position of a ribosyl moiety instead of an OH.
  • a gene is defined to include both transcribed and non-transcribed elements.
  • a gene can include any non- transcribed enhancer and/or promoter (i.e. genomic DNA) that plays a role in determining the level, timing, or tissue specificity of expression of a particular mRNA transcript or non- coding RNA.
  • the 5' UTR, ORF, 3' UTR, introns, as well as non-coding RNAs such as miRNAs, piRNAs, tRNAs, rRNAs, and more, are included as elements of a gene.
  • the hydrophobic modified nucleotide may be an siRNA which includes conventional siRNAs, sd-rxRNAs, asymmetric dsRNAs, single stranded RISC entering polynucleotides, and single stranded RISC inhibiting polynucleotides.
  • aspects of the invention relate to isolated double stranded nucleic acid molecules comprising a guide (antisense) strand and a passenger (sense) strand.
  • double- stranded refers to one or more nucleic acid molecules in which at least a portion of the nucleomonomers are complementary and hydrogen bond to form a double- stranded region.
  • duplex includes the region of the double- stranded nucleic acid molecule(s) that is (are) hydrogen bonded to a complementary sequence.
  • Double-stranded oligonucleotides of the invention may comprise a nucleotide sequence that is sense to a target gene and a complementary sequence that is antisense to the target gene.
  • the sense and antisense nucleotide sequences correspond to the target gene sequence, e.g. , are identical or are sufficiently identical to effect target gene inhibition (e.g.
  • guide strand refers to a polynucleotide or region of a polynucleotide that is substantially (i.e., 80% or more) or 90% complementary to a target nucleic acid of interest and is capable of efficient loading into the RISC complex.
  • a guide strand may be single stranded or part of a duplex and may be comprised of a polynucleotide region that is RNA, DNA or chimeric RNA/DNA.
  • an antisense strand may be complementary, in whole or in part, to a molecule of messenger RNA, an RNA sequence that is not mRNA or sequence of DNA that is either coding or non-coding.
  • the guide strand can be modified with a diverse group of small molecules and/or conjugates and one of the embodiments is related to use of chemical modifications, which improve activity of single stranded guide strands.
  • the phrase "passenger strand” or “sense strand” refers to a polynucleotide or region that has the same nucleotide sequence, in whole or in part, as a target nucleic acid such as a messenger RNA or a sequence of DNA.
  • a sequence is provided, by convention, unless otherwise indicated, it is the sense strand (or region), and the presence of the complementary antisense strand (or region) is implicit.
  • the passenger strand is a second non-essential part of the duplex responsible for promotion of RISC entry and the one, which will be lost after initial RISC loading.
  • double- stranded RNA may be formulated according to the methods of the invention.
  • the invention provides a dsRNA molecule such as a conventional siRNA.
  • Conventional ds siRNA can include a duplex structure of between 18 and 25 basepairs (e.g., 21 base pairs).
  • the dsRNAs include at least one strand that is at least 21 nt long.
  • the dsRNAs include at least one strand that is at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides.
  • the dsRNA has a single-stranded nucleotide overhang of 1 to 4, generally 1 or 2 nucleotides.
  • the single-stranded overhang is located at the 3'-terminal end of the antisense strand or, alternatively, at the 3'- terminal end of the sense strand.
  • the dsRNA may also have a blunt end, generally located at the 5 '-end of the antisense strand.
  • the dsRNA may be cross-linked in some embodiments.
  • Chemical linking of the two separate dsRNA strands may be achieved by any of a variety of well-known techniques, for example by introducing covalent, ionic or hydrogen bonds; hydrophobic interactions, van der Waals or stacking interactions; by means of metal-ion coordination, or through use of purine analogues.
  • Such chemically linked dsRNAs are suitable for packaging in the association complexes described herein.
  • the chemical groups that can be used to modify the dsRNA include, without limitation, methylene blue; bifunctional groups, generally bis-(2- chloroethyl)amine; N-acetyl-N'-(p-glyoxylbenzoyl)cystamine; 4-thiouracil; and psoralen.
  • the linker is a hexa-ethylene glycol linker.
  • the dsRNA are produced by solid phase synthesis and the hexa-ethylene glycol linker is incorporated according to standard methods (e.g., Williams, D. J., and K. B. Hall, Biochem. (1996) 35:14665-14670).
  • the 5'-end of the antisense strand and the 3'- end of the sense strand are chemically linked via a hexaethylene glycol linker.
  • at least one nucleotide of the dsRNA comprises a phosphorothioate or phosphorodithioate groups.
  • the chemical bond at the ends of the dsRNA is generally formed by triple-helix bonds.
  • dsRNA formulated according to the invention also includes sd-rxRNA.
  • sd-rxRNA refers to a class of "self-delivering" RNA molecules described in and incorporated by reference from PCT Publication No. WO 2010/033247 (Application No.
  • sd-rxRNA are asymmetric nucleic acid molecules with a double stranded region of a minimal length such as 8-14, or 8-15, nucleotides, are effective in silencing gene expression. Molecules with such a short double stranded region have not previously been demonstrated to be effective in mediating RNA interference. It had previously been assumed that that there must be a double stranded region of 19 nucleotides or greater. sd-rxRNA molecules can be optimized through chemical modification, and in some instances through attachment of hydrophobic conjugates.
  • RNAi molecules are highly efficient in silencing of target gene expression and offer significant advantages over previously described RNAi molecules including high activity in the presence of serum, efficient self delivery, compatibility with a wide variety of linkers, and reduced presence or complete absence of chemical modifications that are associated with toxicity.
  • an sd-rxRNA molecule comprises a guide strand and a passenger strand, wherein the region of the molecule that is double stranded is from 8-15 nucleotides long, wherein the guide strand contains a single stranded region that is 4-12 nucleotides long, wherein the single stranded region of the guide strand contains 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 phosphorothioate modifications, and wherein at least 40% of the nucleotides of the double stranded nucleic acid are modified.
  • dsRNA formulated according to the invention also includes rxRNAori.
  • rxRNAori refers to a class of RNA molecules described in and incorporated by reference from PCT Publication No. WO2009/102427 (Application No. PCT/US2009/000852), filed on February 11, 2009, and entitled, "MODIFIED RNAI POLYNUCLEOTIDES AND USES THEREOF.”
  • an rxRNAori molecule comprises a double- stranded RNA
  • dsRNA construct of 12-35 nucleotides in length, for inhibiting expression of a target gene, comprising: a sense strand having a 5'-end and a 3'-end, wherein the sense strand is highly modified with 2'-modified ribose sugars, and wherein 3-6 nucleotides in the central portion of the sense strand are not modified with 2'-modified ribose sugars and, an antisense strand having a 5'-end and a 3'-end, which hybridizes to the sense strand and to mRNA of the target gene, wherein the dsRNA inhibits expression of the target gene in a sequence-dependent manner.
  • RNAi-mediated or antisense mediated reduction in gene expression is defined as an RNAi- mediated or antisense mediated reduction in gene expression that can be measured by any number of methods including PCR-based methods, Northern blot analysis, Branched DNA, western blot analysis, and other art recognized techniques.
  • siRNA and the phrase “short interfering RNA” refer to unimolecular nucleic acids and to nucleic acids comprised of two separate strands that are capable of performing RNAi and that have a duplex region that is from about 18 to about 30 base pairs in length.
  • siRNA and the phrase “short interfering RNA” include nucleic acids that also contain moieties other than ribonucleotide moieties, including, but not limited to, modified nucleotides, modified internucleotide linkages, non-nucleotides, deoxynucleotides and analogs of the
  • RNA interference and the term “RNAi” are synonymous and refer to the process by which a single, double, or tripartite molecule (e.g. an siRNA, an shRNA, an miRNA, a piRNA) exerts an effect on a biological process by interacting with one or more components of the RNAi pathway including but not limited to Drosha, RISC, Dicer, etc.
  • the process includes, but is not limited to, gene silencing by degrading mRNA, attenuating translation, interactions with tRNA, rRNA, hnRNA, cDNA and genomic DNA, inhibition of as well as methylation of DNA with ancillary proteins.
  • molecules that modulate RNAi e.g. siRNA, piRNA, or miRNA inhibitors
  • are included in the list of molecules that enhance the RNAi pathway Tomari, Y. et al. Genes Dev. 2005, 19(5):517-29).
  • the double- stranded oligonucleotide of the invention is double-stranded over its entire length, i.e. , with no overhanging single- stranded sequence at either end of the molecule, i.e., is blunt-ended.
  • the individual nucleic acid molecules can be of different lengths.
  • a double- stranded oligonucleotide of the invention is not double-stranded over its entire length. For instance, when two separate nucleic acid molecules are used, one of the molecules, e.g.
  • the first molecule comprising an antisense sequence can be longer than the second molecule hybridizing thereto (leaving a portion of the molecule single- stranded).
  • the term "mismatch” includes a situation in where Watson-Crick base pairing does not take place between a nucleotide of a sense strand and a nucleotide of an antisense strand.
  • mismatch would be an A across from a G, a C across from an A, a U across from a C, an A across from an A, a G across from a G, a C across from a C, and so on.
  • Mismatches are also meant to include an basic residue across from a nucleotide or modified nucleotide, an acyclic residue across from a nucleotide or modified nucleotide, a gap, or an unpaired loop.
  • a mismatch as used herein includes any alteration at a given position which decreases the thermodynamic stability at or in the vicinity of the position where the alteration appears, such that the thermodynamic stability of the duplex at the particular position is less than the
  • mismatches include a G across from an A, and an A across from a C.
  • a particularly preferred mismatch comprises an A across from an A, G across from a G, C across from a C, and U across from a U.
  • a double-stranded oligonucleotide of the invention contains mismatches and/or loops or bulges, but is double- stranded over at least about 70% of the length of the oligonucleotide. In another embodiment, a double-stranded oligonucleotide of the invention is double-stranded over at least about 80% of the length of the oligonucleotide. In another embodiment, a double- stranded oligonucleotide of the invention is double- stranded over at least about 90%-95% of the length of the oligonucleotide.
  • a double- stranded oligonucleotide of the invention is double-stranded over at least about 96%-98% of the length of the oligonucleotide.
  • the double-stranded oligonucleotide of the invention contains at least or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mismatches.
  • the term "complementary” refers to the ability of polynucleotides to form base pairs with one another.
  • Base pairs are typically formed by hydrogen bonds between nucleotide units in antiparallel polynucleotide strands or regions.
  • Complementary polynucleotide strands or regions can base pair in the Watson-Crick manner (e.g., A to T, A to U, C to G, G to U), or in any other manner that allows for the formation of stable duplexes.
  • the guide strand has complementarity to a target gene. Complementarity between the guide strand and the target gene may exist over any portion of the guide strand.
  • Complementarity as used herein may be perfect complementarity or less than perfect complementarity as long as the guide strand is sufficiently complementary to the target that it mediates RNAi. In some embodiments complementarity refers to less than 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% mismatch between the guide strand and the target. Perfect complementarity refers to 100% complementarity. Perfect complementarity refers to the situation in which each nucleotide unit of one polynucleotide strand or region can hydrogen bond with each nucleotide unit of a second polynucleotide strand or region. Less than perfect complementarity refers to the situation in which some, but not all, nucleotide units of two strands or two regions can hydrogen bond with each other.
  • the invention has the advantage of being able to tolerate sequence variations that might be expected due to genetic mutation, strain polymorphism, or evolutionary divergence.
  • siRNA sequences with insertions, deletions, and single point mutations relative to the target sequence have also been found to be effective for inhibition.
  • not all positions of a siRNA contribute equally to target recognition. Mismatches in the center of the siRNA are most critical and essentially abolish target RNA cleavage. Mismatches upstream of the center or upstream of the cleavage site referencing the antisense strand are tolerated but significantly reduce target RNA cleavage.
  • Mismatches downstream of the center or cleavage site referencing the antisense strand preferably located near the 3' end of the antisense strand, e.g. 1, 2, 3, 4, 5 or 6 nucleotides from the 3' end of the antisense strand, are tolerated and reduce target RNA cleavage only slightly.
  • duplex polynucleotides In contrast to single- stranded polynucleotides, duplex polynucleotides have been difficult to deliver to a cell as they have rigid structures and a large number of negative charges which makes membrane transfer difficult. Unexpectedly, as described in, and incorporated by reference from, PCT/US2009/005247, it was found that sd-rxRNA molecules, although partially double-stranded, are recognized in vivo as single- stranded and, as such, are capable of efficiently being delivered across cell membranes. As a result, these polynucleotides are capable in many instances of self delivery.
  • the polynucleotides of the invention may be formulated in a manner similar to conventional RNAi agents or they may be delivered to the cell or subject alone (or with non-delivery type carriers) and allowed to self deliver.
  • self delivering asymmetric double-stranded RNA molecules are provided in which one portion of the molecule resembles a conventional RNA duplex and a second portion of the molecule is single stranded.
  • a longer duplex polynucleotide including a first polynucleotide that ranges in size from about 16 to about 30 nucleotides; a second polynucleotide that ranges in size from about 26 to about 46 nucleotides, wherein the first polynucleotide (the antisense strand) is complementary to both the second polynucleotide (the sense strand) and a target gene, and wherein both polynucleotides form a duplex and wherein the first polynucleotide contains a single stranded region longer than 6 bases in length and is modified with alternative chemical modification pattern, and/or includes a conjugate moiety that facilitates cellular delivery.
  • between about 40% to about 90% of the nucleotides of the passenger strand between about 40% to about 90% of the nucleotides of the guide strand, and between about 40% to about 90% of the nucleotides of the single stranded region of the first polynucleotide are chemically modified nucleotides.
  • the chemically modified nucleotide in the polynucleotide duplex may be any chemically modified nucleotide known in the art, such as those discussed in detail above.
  • the chemically modified nucleotide is selected from the group consisting of 2' F modified nucleotides, 2'-0-methyl modified and 2'deoxy nucleotides.
  • the chemically modified nucleotides results from "hydrophobic modifications" of the nucleotide base.
  • the chemically modified nucleotides are phosphorothioates.
  • chemically modified nucleotides are combination of phosphorothioates, 2'-0- methyl, 2'deoxy, hydrophobic modifications and phosphorothioates.
  • these groups of modifications refer to modification of the ribose ring, back bone and nucleotide, it is feasible that some modified nucleotides will carry a combination of all three modification types.
  • At least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the nucleotides are modified.
  • the chemical modification is not the same across the various regions of the duplex.
  • the first polynucleotide (the passenger strand), has a large number of diverse chemical modifications in various positions. For this polynucleotide up to 90% of nucleotides might be chemically modified and/or have mismatches introduced.
  • Single stranded modified and non modified RNA, DNA molecules may also be formulated according to the methods of the invention.
  • An example of a single stranded polynucleotide that may be formulated according to the invention is those ODN described in, and incorporated by reference from, PCT/US2009/004326, filed on July 23, 2009, and entitled "RNAi Constructs and Uses Thereof.” Briefly, these ssRNA can form double stranded structures based on internal interactions or on interactions with identical sequences.
  • they may include two identical single-stranded polynucleotides, each of the single- stranded polynucleotide comprising a 5 '-stem sequence having a 5 '-end, a 3 '-stem sequence having a 3 '-end, and a linker sequence linking the 5 '-stem sequence and the 3 '-stem sequence, wherein: (1) the 5 '-stem sequence of a first single-stranded polynucleotide hybridize with the 3 '-stem sequence of a second single-stranded polynucleotide to form a first double-stranded stem region; (2) the 5 '-stem sequence of the second single-stranded polynucleotide hybridize with the 3 '-stem sequence of the first single- stranded
  • the linker sequences of the first and the second single-stranded polynucleotides form a loop or bulge connecting the first and the second double-stranded stem regions, wherein the 5'-stem sequence and at least a portion of the linker sequence form a guide sequence complementary to a transcript (such as an mRNA or a non-coding RNA) of a target gene.
  • a transcript such as an mRNA or a non-coding RNA
  • Single stranded RNA molecules also include for instance microRNAs (miRNAs).
  • miRNAs are small noncoding RNA molecules that are capable of causing post- transcriptional silencing of specific genes in cells such as by the inhibition of translation or through degradation of the targeted mRNA.
  • a miRNA can be completely complementary or can have a region of noncomplementarity with a target nucleic acid, consequently resulting in a "bulge" at the region of non-complementarity.
  • the region of noncomplementarity (the bulge) can be flanked by regions of sufficient complementarity, preferably complete complementarity to allow duplex formation.
  • the regions of complementarity are at least 8 to 10 nucleotides long (e.g., 8, 9, or 10 nucleotides long).
  • a miRNA can inhibit gene expression by repressing translation, such as when the microRNA is not completely complementary to the target nucleic acid, or by causing target RNA degradation, which is believed to occur only when the miRNA binds its target with perfect complementarity.
  • the invention also can include double-stranded precursors of miRNAs that may or may not form a bulge when bound to their targets.
  • a miRNA or pre-miRNA can be 16-100 nucleotides in length, and more preferably from 16-80 nucleotides in length.
  • Mature miRNAs can have a length of 16-30 nucleotides, preferably 21-25 nucleotides, particularly 21, 22, 23, 24, or 25 nucleotides.
  • MicroRNA precursors can have a length of 70-100 nucleotides and have a hairpin conformation.
  • MicroRNAs can be generated in vivo from pre-miRNAs by enzymes called Dicer and Drosha that specifically process long pre-miRNA into functional miRNA.
  • Single Stranded DNA molecules include for instance antisense-oligonucleotides.
  • the single-stranded oligonucleotides featured in the invention include antisense nucleic acids.
  • An "antisense" nucleic acid includes a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a gene expression product, e.g., complementary to the coding strand of a double- stranded cDNA molecule or complementary to an RNA sequence, e.g., a pre- mRNA, mRNA, miRNA, or pre-miRNA. Accordingly, an antisense nucleic acid can form hydrogen bonds with a sense nucleic acid target.
  • antisense nucleic acids can be designed according to the rules of Watson and Crick base pairing.
  • the antisense nucleic acid molecule can be complementary to a portion of the coding or noncoding region of an RNA, e.g., a pre- mRNA or mRNA.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of a pre-mRNA or mRNA, e.g., the 5' UTR.
  • An antisense oligonucleotide can be, for example, about 10 to 25 nucleotides in length (e.g., 11, 12, 13, 14, 15, 16, 18, 19, 20, 21, 22, 23, or 24 nucleotides in length).
  • An antisense oligonucleotide can also be complementary to a miRNA or pre-miRNA.
  • an antisense nucleic acid can be constructed using chemical synthesis and/or enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense 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 between the antisense and target nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • Other appropriate nucleic acid modifications are described herein.
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest).
  • An antisense agent can include ribonucleotides only, deoxyribonucleotides only (e.g., oligodeoxynucleotides), or both deoxyribonucleotides and ribonucleotides.
  • an antisense agent consisting only of ribonucleotides can hybridize to a complementary RNA, and prevent access of the translation machinery to the target RNA transcript, thereby preventing protein synthesis.
  • An antisense molecule including only deoxyribonucleotides, or deoxyribonucleotides and ribonucleotides, e.g., DNA sequence flanked by RNA sequence at the 5' and 3' ends of the antisense agent, can hybridize to a complementary RNA, and the RNA target can be subsequently cleaved by an enzyme, e.g., RNAse H. Degradation of the target RNA prevents translation.
  • the flanking RNA sequences can include 2'-0-methylated nucleotides, and phosphorothioate linkages, and the internal DNA sequence can include phosphorothioate internucleotide linkages.
  • the internal DNA sequence is preferably at least five nucleotides in length when targeting by RNAseH activity is desired.
  • nucleic acid is a decoy-oligonucleotide, e.g., a decoy RNA.
  • a decoy nucleic acid resembles a natural nucleic acid, but is modified in such a way as to inhibit or interrupt the activity of the natural nucleic acid.
  • a decoy RNA can mimic the natural binding domain for a ligand. The decoy RNA therefore competes with natural binding target for the binding of a specific ligand.
  • the natural binding target can be an endogenous nucleic acid, e.g., a pre-miRNA, miRNA, premRNA, mRNA or DNA.
  • Aptamer are also oligonucleotides which may be formulated according to the methods of the invention.
  • An aptamer binds to a non-nucleic acid ligand, such as a small organic molecule or protein, e.g., a transcription or translation factor, and subsequently modifies (e.g., inhibits) activity.
  • a non-nucleic acid ligand such as a small organic molecule or protein, e.g., a transcription or translation factor
  • An aptamer can fold into a specific structure that directs the recognition of the targeted binding site on the non-nucleic acid ligand.
  • An aptamer can contain any of the modifications described herein.
  • Antagomirs which are single stranded, double stranded, partially double stranded and hairpin structured chemically modified oligonucleotides that target a microRNA, are also useful according to the invention.
  • An antagomir may be for instance at least 8 or more contiguous nucleotides substantially complementary to an endogenous miRNA and more particularly agents that include 12 or more contiguous nucleotides substantially
  • an antagomir featured in the invention includes a nucleotide sequence sufficiently complementary to hybridize to a miRNA target sequence of about 12 to 25 nucleotides, preferably about 15 to 23 nucleotides.
  • An antagomir that is substantially complementary to a nucleotide sequence of an miRNA can be delivered to a cell or a human to inhibit or reduce the activity of an endogenous miRNA, such as when aberrant or undesired miRNA activity, or insufficient activity of a target mRNA that hybridizes to the endogenous miRNA, is linked to a disease or disorder.
  • asymmetric dsRNA refers to a duplex, where the length of one strand is substantially higher then the other. As a result, there is an additional single stranded region extending from a duplex.
  • the chemical modification patterns for different regions of the asymmetric dsRNA can be different.
  • the term "overhang” refers to terminal non-base pairing nucleotide(s) resulting from one strand or region extending beyond the terminus of the complementary strand to which the first strand or region forms a duplex.
  • One or more polynucleotides that are capable of forming a duplex through hydrogen bonding can have overhangs. In a conventional siRNA molecule the overhand length generally doesn't exceed 5 bases in length.
  • duplex refers to a region of double- stranded structure formed by two antiparallel polynucleotide strands as a result of base- pairing between the strands.
  • a duplex may be formed between two separate polynucleotides, or the strands may be contained with a single polynucleotide sequence e.g. a hairpin structure where the "loop" portion of the hairpin allows the two strands to adopt an antiparallel configuration relative to each other.
  • the polynucleotide is unmodified. In other embodiments, at least one nucleotide is modified. In further embodiments, the modification includes a 2'-H or 2 '-modified ribose sugar at the 2nd nucleotide from the 5 '-end of the guide sequence.
  • the "2nd nucleotide” is defined as the second nucleotide from the 5 '-end of the polynucleotide.
  • modification pattern refers to chemical modification pattern, which is found to be optimal for a particular application.
  • the chemical modification pattern enables generalization of chemical principles for many different sequences. Usually, chemical modification pattern is link to easier functional position or both sequence and functional position.
  • the 2'F modification of every C and U in the guide strand of the duplex is considered to be an acceptable chemical modification pattern for a guide strand.
  • Another example, is fully Omethyl modified oligo with several phosphorothioates on the ends is an acceptable chemical modification pattern for miRNA inhibitors.
  • nucleotides of the invention may be modified at various locations, including the sugar moiety, the phosphodiester linkage, and/or the base.
  • Sugar moieties include natural, unmodified sugars, e.g. , monosaccharide (such as pentose, e.g. , ribose, deoxyribose), modified sugars and sugar analogs.
  • monosaccharide such as pentose, e.g. , ribose, deoxyribose
  • possible modifications of nucleomonomers, particularly of a sugar moiety include, for example, replacement of one or more of the hydroxyl groups with a halogen, a heteroatom, an aliphatic group, or the functionalization of the hydroxyl group as an ether, an amine, a thiol, or the like.
  • modified nucleomonomers are 2'-0-methyl nucleotides. Such 2'-0-methyl nucleotides may be referred to as "methylated,” and the corresponding nucleotides may be made from unmethylated nucleotides followed by alkylation or directly from methylated nucleotide reagents. Modified nucleomonomers may be used in combination with unmodified nucleomonomers. For example, an oligonucleotide of the invention may contain both methylated and unmethylated nucleomonomers.
  • modified nucleomonomers include sugar- or backbone-modified ribonucleotides.
  • Modified ribonucleotides may contain a non-naturally occurring base (instead of a naturally occurring base), such as uridines or cytidines modified at the 5'- position, e.g. , 5'-(2-amino)propyl uridine and 5'-bromo uridine; adenosines and guanosines modified at the 8-position, e.g. , 8-bromo guanosine; deaza nucleotides, e.g.
  • sugar-modified ribonucleotides may have the 2' -OH group replaced by a H, alkoxy (or OR), R or alkyl, halogen, SH, SR, amino (such as N3 ⁇ 4, NHR, NR 2, ), or CN group, wherein R is lower alkyl, alkenyl, or alkynyl.
  • Modified ribonucleotides may also have the phosphodiester group connecting to adjacent ribonucleotides replaced by a modified group, e.g., of phosphorothioate group. More generally, the various nucleotide modifications may be combined.
  • the antisense (guide) strand may be substantially identical to at least a portion of the target gene (or genes), at least with respect to the base pairing properties, the sequence need not be perfectly identical to be useful, e.g. , to inhibit expression of a target gene's phenotype. Generally, higher homology can be used to compensate for the use of a shorter antisense gene. In some cases, the antisense strand generally will be substantially identical (although in antisense orientation) to the target gene.
  • RNA having 2'-0-methyl nucleomonomers may not be recognized by cellular machinery that is thought to recognize unmodified RNA.
  • the use of 2'-0-methylated or partially 2'-0-methylated RNA may avoid the interferon response to double- stranded nucleic acids, while maintaining target RNA inhibition. This may be useful, for example, for avoiding the interferon or other cellular stress responses, both in short RNAi (e.g. , siRNA) sequences that induce the interferon response, and in longer RNAi sequences that may induce the interferon response.
  • the sugar moiety can be a hexose and incorporated into an oligonucleotide as described (Augustyns, K., et al., Nucl. Acids. Res. 18:4711 (1992)).
  • Exemplary nucleomonomers can be found, e.g. , in U.S. Pat. No. 5,849,902, incorporated by reference herein.
  • the nucleomonomers of an oligonucleotide of the invention are RNA nucleotides.
  • the nucleomonomers of an oligonucleotide of the invention are modified RNA nucleotides.
  • the oligonucleotides contain modified RNA nucleotides.
  • nucleoside includes bases which are covalently attached to a sugar moiety, preferably ribose or deoxyribose.
  • examples of preferred nucleosides include ribonucleosides and deoxyribonucleosides.
  • Nucleosides also include bases linked to amino acids or amino acid analogs which may comprise free carboxyl groups, free amino groups, or protecting groups. Suitable protecting groups are well known in the art (see P. G. M. Wuts and T. W. Greene, "Protective Groups in Organic Synthesis", 2 nd Ed., Wiley-Interscience, New York, 1999).
  • nucleotide includes nucleosides which further comprise a phosphate group or a phosphate analog.
  • linkage includes a naturally occurring, unmodified phosphodiester moiety (-0-(P0 2 ⁇ )-0-) that covalently couples adjacent nucleomonomers.
  • substitute linkage includes any analog or derivative of the native phosphodiester group that covalently couples adjacent nucleomonomers. Substitute linkages include phosphodiester analogs, e.g. , phosphorothioate, phosphorodithioate, and P- ethyoxyphosphodiester, P-ethoxyphosphodiester, P-alkyloxyphosphotriester,
  • nonphosphorus containing linkages e.g. , acetals and amides.
  • linkages e.g., Bjergarde et al. 1991. Nucleic Acids Res. 19:5843; Caruthers et al. 1991. Nucleosides Nucleotides. 10:47).
  • non-hydrolizable linkages are preferred, such as phosphorothiate linkages.
  • conjugates that can be attached to the end (3' or 5' end), the loop region, or any other parts of the miniRNA might include a sterol, sterol type molecule, peptide, small molecule, protein, etc.
  • a miniRNA may contain more than one conjugate (same or different chemical nature).
  • the conjugate is cholesterol.
  • Another way to increase target gene specificity, or to reduce off-target silencing effect is to introduce a 2' -modification (such as the 2'-0 methyl modification) at a position corresponding to the second 5 ' -end nucleotide of the guide sequence.
  • This allows the positioning of this 2 '-modification in the Dicer-resistant hairpin structure, thus enabling one to design better RNAi constructs with less or no off-target silencing.
  • a hairpin polynucleotide of the invention can comprise one nucleic acid portion which is DNA and one nucleic acid portion which is RNA.
  • Antisense (guide) sequences of the invention can be "chimeric oligonucleotides" which comprise an RNA-like and a DNA-like region.
  • At least a portion of the contiguous polynucleotides are linked by a substitute linkage, e.g., a. phosphorothioate linkage.
  • nucleotides beyond the guide sequence (2'- modified or not) are linked by phosphorothioate linkages. Such constructs tend to have improved pharmacokinetics due to their higher affinity for serum proteins.
  • phosphorothioate linkages in the non-guide sequence portion of the polynucleotide generally do not interfere with guide strand activity, once the latter is loaded into RISC.
  • polynucleotides have been shown to be active in loading into RISC and inducing gene silencing. However, the level of activity for single stranded polynucleotides appears to be 2 to 4 orders of magnitude lower when compared to a duplex polynucleotide.
  • the present invention provides a description of the chemical modification patterns, which may (a) significantly increase stability of the single stranded polynucleotide (b) promote efficient recognition of the polynucleotide by the RISC as a substrate and/or loading of the polynucleotide into the RISC complex and (c) improve uptake of the single stranded nucleotide by the cell.
  • the chemical modification patterns may include combination of ribose, backbone, hydrophobic nucleoside and conjugate type of modifications.
  • the 5' end of the single polynucleotide may be chemically phosphorylated.
  • the present invention provides a description of the chemical modifications patterns, which improve functionality of RISC inhibiting
  • polynucleotides Single stranded polynucleotides have been shown to inhibit activity of a preloaded RISC complex through the substrate competition mechanism.
  • antagomers the activity usually requires high concentration and in vivo delivery is not very effective.
  • polynucleotides This includes single stranded RISC entering polynucleotides, single stranded RISC inhibiting polynucleotides, conventional duplexed polynucleotides of variable length (15- 40 bp), asymmetric duplexed polynucleotides, and the like.
  • Polynucleotides may be modified with a wide variety of chemical modification patterns, including 5' end, ribose, backbone and hydrophobic nucleoside modifications.
  • the modified RNA polynucleotide of the invention with the above -referenced 5'-end modification exhibits significantly (e.g. , at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more) less "off- target" gene silencing when compared to similar constructs without the specified 5 '-end modification, thus greatly improving the overall specificity of the RNAi reagent or therapeutics.
  • modified bases refer to nucleotide bases such as, for example, adenine, guanine, cytosine, thymine, and uracil, xanthine, inosine, and queuosine that have been modified by the replacement or addition of one or more atoms or groups.
  • nucleotide bases such as, for example, adenine, guanine, cytosine, thymine, and uracil, xanthine, inosine, and queuosine that have been modified by the replacement or addition of one or more atoms or groups.
  • modifications that can comprise nucleotides that are modified with respect to the base moieties include but are not limited to, alkylated, halogenated, thiolated, aminated, amidated, or acetylated bases, in various combinations.
  • More specific modified bases include, for example, 5-propynyluridine, 5-propynylcytidine, 6- methyladenine, 6-methylguanine, ⁇ , ⁇ ,-dimethyladenine, 2-propyladenine, 2-propylguanine, 2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine and other nucleotides having a modification at the 5 position, 5-(2-amino)propyl uridine, 5- halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 3- methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine, 2,2- dimethylguanosine, 5-methylaminoethyluridine, 5-methyloxyuridine, deazanucleotides such as 7-deaza-adenosine, 6-azouridine, 6-azocytidine
  • Modified nucleotides also include those nucleotides that are modified with respect to the sugar moiety, as well as nucleotides having sugars or analogs thereof that are not ribosyl.
  • the sugar moieties may be, or be based on, mannoses, arabinoses, glucopyranoses,
  • nucleotide is also meant to include what are known in the art as universal bases.
  • universal bases include but are not limited to 3-nitropyrrole, 5-nitroindole, or nebularine.
  • chemical modifications of the first or second polynucleotide include, but not limited to, 5' position modification of Uridine and Cytosine (4-pyridyl, 2- pyridyl, indolyl, phenyl (C 6 H 5 OH); tryptophanyl (C8H6N)CH2CH(NH2)CO), isobutyl, butyl, aminobenzyl; phenyl; naphthyl, etc), where the chemical modification might alter base pairing capabilities of a nucleotide.
  • 5' position modification of Uridine and Cytosine (4-pyridyl, 2- pyridyl, indolyl, phenyl (C 6 H 5 OH); tryptophanyl (C8H6N)CH2CH(NH2)CO), isobutyl, butyl, aminobenzyl; phenyl; naphthyl, etc), where the chemical modification might alter base pairing capabilities of a nucleotide.
  • a unique feature of this aspect of the invention involves the use of hydrophobic modification on the bases.
  • the hydrophobic modifications are preferably positioned near the 5' end of the guide strand, in other embodiments, they localized in the middle of the guides strand, in other embodiment they localized at the 3 ' end of the guide strand and yet in another embodiment they are distributed thought the whole length of the polynucleotide.
  • the same type of patterns is applicable to the passenger strand of the duplex.
  • the other part of the molecule is a single stranded region.
  • the single stranded region ranges from 2-40 nucleotides.
  • the single stranded region can be 2, 3, 4, 5 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides.
  • the single stranded region can range from 7 to 40 nucleotides.
  • the single stranded region of the first polynucleotide contains modifications selected from the group consisting of between 40% and 90% hydrophobic base modifications, between 40%-90% phosphorothioates, between 40% -90% modification of the ribose moiety, and any combination of the preceding.
  • the duplex polynucleotide includes a mismatch between nucleotide 9, 11, 12, 13, or 14 on the guide strand (first polynucleotide) and the opposite nucleotide on the sense strand (second polynucleotide) to promote efficient guide strand loading.
  • Oligonucleotides of the invention can be synthesized by any method known in the art, e.g., using enzymatic synthesis and/or chemical synthesis.
  • the oligonucleotides can be synthesized in vitro (e.g. , using enzymatic synthesis and chemical synthesis) or in vivo (using recombinant DNA technology well known in the art).
  • Oligonucleotides and oligonucleotide compositions are contacted with (i.e., brought into contact with, also referred to herein as administered or delivered to) and taken up by one or more cells or a cell lysate.
  • the term "cells” includes prokaryotic and eukaryotic cells, preferably vertebrate cells, and, more preferably, mammalian cells. In a preferred
  • the oligonucleotide compositions of the invention are contacted with human cells.
  • Oligonucleotide compositions of the invention can be contacted with cells in vitro, e.g., in a test tube or culture dish, (and may or may not be introduced into a subject) or in vivo, e.g., in a subject such as a mammalian subject. Oligonucleotides are taken up by cells at a slow rate by endocytosis, but endocytosed oligonucleotides are generally sequestered and not available, e.g. , for hybridization to a target nucleic acid molecule. In one embodiment, cellular uptake can be facilitated by electroporation or calcium phosphate precipitation.
  • RNA molecules of the invention can be delivered by using various beta-glucan containing particles.
  • the beta-glucan component of the particle may be similar to the beta- glucan material described in US 2005/0281781 Al, WO 2006/007372, and WO 2007/050643 (all incorporated herein by reference) and discussed further above.
  • the beta-glucan particle is derived from yeast.
  • the glucan particle may contain a polymer, such as those with a molecular weight of at least about 1000 Da, 10,000 Da, 50,000 Da, 100 kDa, 500 kDa, etc.
  • Preferred polymers include (without limitation) cationic polymers, chitosans, or PEI (polyethylenimine), etc.
  • Such beta-glucan based delivery system may be formulated for oral delivery, where the orally delivered beta-glucan /RNA complexes may be engulfed by macrophages or other related phagocytic cells, which may in turn release the RNA molecules in selected in vivo sites.
  • the RNA molecules may changes the expression of certain macrophage target genes.
  • the optimal protocol for uptake of oligonucleotides will depend upon a number of factors, the most crucial being the type of cells that are being used. Other factors that are important in uptake include, but are not limited to, the nature and concentration of the oligonucleotide, the confluence of the cells, the type of culture the cells are in (e.g. , a suspension culture or plated) and the type of media in which the cells are grown.
  • oligonucleotides can also be improved by targeting the
  • the targeting moieties can be conjugated to the oligonucleotides or attached to a carrier group (i.e. , poly(L- lysine) or liposomes) linked to the oligonucleotides. This method is well suited to cells that display specific receptor-mediated endocytosis.
  • oligonucleotide conjugates to 6-phosphomannosylated proteins are internalized 20-fold more efficiently by cells expressing mannose 6-phosphate specific receptors than free oligonucleotides.
  • the oligonucleotides may also be coupled to a ligand for a cellular receptor using a biodegradable linker.
  • the delivery construct is mannosylated streptavidin which forms a tight complex with biotinylated oligonucleotides.
  • Mannosylated streptavidin was found to increase 20-fold the internalization of biotinylated oligonucleotides. (Vlassov et al. 1994. Biochimica et Biophysica Acta 1197:95-108).
  • the optimal course of administration or delivery of the oligonucleotides may vary depending upon the desired result and/or on the subject to be treated.
  • administration refers to contacting cells with oligonucleotides and can be performed in vitro or in vivo.
  • the dosage of oligonucleotides may be adjusted to optimally reduce expression of a protein translated from a target nucleic acid molecule, e.g. , as measured by a readout of RNA stability or by a therapeutic response, without undue experimentation.
  • expression of the protein encoded by the nucleic acid target can be measured to determine whether or not the dosage regimen needs to be adjusted accordingly.
  • an increase or decrease in RNA or protein levels in a cell or produced by a cell can be measured using any art recognized technique. By determining whether transcription has been decreased, the effectiveness of the oligonucleotide in inducing the cleavage of a target RNA can be determined.
  • oligonucleotide compositions can be used alone or in conjunction with a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes appropriate solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, it can be used in the therapeutic compositions. Supplementary active ingredients can also be incorporated into the compositions.
  • the formulations of the present invention can be administered to a subject in a variety of forms adapted to the chosen route of administration, e.g., parenterally, orally, or intraperitoneally.
  • Parenteral administration includes
  • administration by the following routes: intravenous; intramuscular; interstitially;
  • intraarterially subcutaneous; intra ocular; intrasynovial; trans epithelial, including transdermal; pulmonary via inhalation; ophthalmic; sublingual and buccal; topically, including ophthalmic; dermal; ocular; rectal; and nasal inhalation via insufflation.
  • compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble or water-dispersible form.
  • suspensions of the active compounds as appropriate oily injection suspensions may be administered.
  • Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, or dextran, optionally, the suspension may also contain stabilizers.
  • the oligonucleotides of the invention can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution.
  • the oligonucleotides may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included in the invention.
  • compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets.
  • thickeners, flavoring agents, diluents, emulsifiers, dispersing aids, or binders may be used in pharmaceutical preparations for oral administration.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives, and detergents.
  • Transmucosal administration may be through nasal sprays or using suppositories.
  • the oligonucleotides are formulated into conventional oral administration forms such as capsules, tablets, and tonics.
  • the oligonucleotides of the invention are formulated into ointments, salves, gels, or creams as known in the art.
  • Drug delivery vehicles can be chosen e.g. , for in vitro, for systemic, or for topical administration. These vehicles can be designed to serve as a slow release reservoir or to deliver their contents directly to the target cell.
  • An advantage of using some direct delivery drug vehicles is that multiple molecules are delivered per uptake. Such vehicles have been shown to increase the circulation half-life of drugs that would otherwise be rapidly cleared from the blood stream.
  • Some examples of such specialized drug delivery vehicles which fall into this category are liposomes, biodegradable nanocapsules, and bioadhesive microspheres.
  • the described oligonucleotides may be administered systemically to a subject.
  • Systemic absorption refers to the entry of drugs into the blood stream followed by distribution throughout the entire body.
  • Administration routes which lead to systemic absorption include: intravenous, subcutaneous, intraperitoneal, and intranasal. Each of these administration routes delivers the oligonucleotide to accessible diseased cells. Following subcutaneous administration, the therapeutic agent drains into local lymph nodes and proceeds through the lymphatic network into the circulation. The rate of entry into the circulation has been shown to be a function of molecular weight or size.
  • compositions of oligonucleotides are formulated as microemulsions.
  • a microemulsion is a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution.
  • microemulsions are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a 4th component, generally an intermediate chain-length alcohol to form a transparent system.
  • Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs.
  • Lipid based microemulsions both oil/water and water/oil have been proposed to enhance the oral bioavailability of drugs.
  • Microemulsions offer improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (Constantinides et al., Pharmaceutical Research, 1994, 11 : 1385; Ho et al., J. Pharm. Sci., 1996, 85: 138-143). Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications.
  • microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of oligonucleotides from the gastrointestinal tract, as well as improve the local cellular uptake of oligonucleotides within the gastrointestinal tract, vagina, buccal cavity and other areas of administration.
  • the present invention employs various penetration enhancers to affect the efficient delivery of nucleic acids, particularly oligonucleotides, to the skin of animals.
  • nucleic acids particularly oligonucleotides
  • the present invention employs various penetration enhancers to affect the efficient delivery of nucleic acids, particularly oligonucleotides, to the skin of animals.
  • non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer.
  • penetration enhancers also act to enhance the permeability of lipophilic drugs.
  • Five categories of penetration enhancers that may be used in the present invention include: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non- surfactants.
  • Other agents may be utilized to enhance the penetration of the administered oligonucleotides include: glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-15 pyrrol, azones, and terpenes such as limonene, and menthone.
  • the oligonucleotides, especially in lipid formulations, can also be administered by coating a medical device, for example, a catheter, such as an angioplasty balloon catheter, with a cationic lipid formulation. Coating may be achieved, for example, by dipping the medical device into a lipid formulation or a mixture of a lipid formulation and a suitable solvent, for example, an aqueous-based buffer, an aqueous solvent, ethanol, methylene chloride, chloroform and the like. An amount of the formulation will naturally adhere to the surface of the device which is subsequently administered to a subject, as appropriate.
  • a medical device for example, a catheter, such as an angioplasty balloon catheter
  • Coating may be achieved, for example, by dipping the medical device into a lipid formulation or a mixture of a lipid formulation and a suitable solvent, for example, an aqueous-based buffer, an aqueous solvent, ethanol, methylene chloride, chloroform and the like.
  • a lyophilized mixture of a lipid formulation may be specifically bound to the surface of the device.
  • binding techniques are described, for example, in K. Ishihara et al., Journal of Biomedical Materials Research, Vol. 27, pp. 1309-1314 (1993), the disclosures of which are incorporated herein by reference in their entirety.
  • fatty acids, sterols and oils can be used to effectively load glucan shells with oligonucleotide components.
  • the content of a hydrophobic mix can be further optimized to promote not only loading but dissociation and endosomal escape upon change of pH after cellular internalization.
  • fatty acids with pK values between approximately 4.5-6.5 might provide an additional advantage.
  • amphiphilic molecules such as amphiphilic peptides can also be used. As discussed in the Examples section, ratios of compounds can be optimized to provide optimal balance between particle loading and endosomal escape
  • the useful dosage to be administered and the particular mode of administration will vary depending upon such factors as the cell type, or for in vivo use, the age, weight and the particular animal and region thereof to be treated, the particular oligonucleotide and delivery method used, the therapeutic or diagnostic use contemplated, and the form of the formulation, for example, suspension, emulsion, micelle or liposome, as will be readily apparent to those skilled in the art.
  • dosage is administered at lower levels and increased until the desired effect is achieved.
  • the amount of lipid compound that is administered can vary and generally depends upon the amount of oligonucleotide agent being administered.
  • the weight ratio of lipid compound to oligonucleotide agent is preferably from about 1: 1 to about 15: 1, with a weight ratio of about 5: 1 to about 10: 1 being more preferred.
  • the amount of cationic lipid compound which is administered will vary from between about 0.1 milligram (mg) to about 1 gram (g).
  • mg milligram
  • g 1 gram
  • the agents of the invention are administered to subjects or contacted with cells in a biologically compatible form suitable for pharmaceutical administration.
  • biologically compatible form suitable for administration is meant that the oligonucleotide is administered in a form in which any toxic effects are outweighed by the therapeutic effects of the oligonucleotide.
  • oligonucleotides can be administered to subjects.
  • subjects include mammals, e.g. , humans and other primates; cows, pigs, horses, and farming (agricultural) animals; dogs, cats, and other domesticated pets; mice, rats, and transgenic non-human animals.
  • mammals e.g. , humans and other primates
  • dogs, cats, and other domesticated pets mice, rats, and transgenic non-human animals.
  • an active amount of an oligonucleotide of the present invention is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result.
  • an active amount of an oligonucleotide may vary according to factors such as the type of cell, the oligonucleotide used, and for in vivo uses the disease state, age, sex, and weight of the individual, and the ability of the oligonucleotide to elicit a desired response in the individual.
  • Establishment of therapeutic levels of oligonucleotides within the cell is dependent upon the rates of uptake and efflux or degradation. Decreasing the degree of degradation prolongs the intracellular half-life of the oligonucleotide.
  • chemically- modified oligonucleotides e.g. , with modification of the phosphate backbone, may require different dosing.
  • oligonucleotide and number of doses administered will depend upon the data generated experimentally and in clinical trials. Several factors such as the desired effect, the delivery vehicle, disease indication, and the route of administration, will affect the dosage. Dosages can be readily determined by one of ordinary skill in the art and formulated into the subject pharmaceutical compositions. Preferably, the duration of treatment will extend at least through the course of the disease symptoms.
  • Dosage regimen may be adjusted to provide the optimum therapeutic response.
  • the oligonucleotide may be repeatedly administered, e.g. , several doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • One of ordinary skill in the art will readily be able to determine appropriate doses and schedules of administration of the subject oligonucleotides, whether the oligonucleotides are to be administered to cells or to subjects.
  • nucleic acids Physical methods of introducing nucleic acids include injection of a solution containing the nucleic acid, bombardment by particles covered by the nucleic acid, soaking the cell or organism in a solution of the nucleic acid, or electroporation of cell membranes in the presence of the nucleic acid.
  • a viral construct packaged into a viral particle would accomplish both efficient introduction of an expression construct into the cell and transcription of nucleic acid encoded by the expression construct.
  • Other methods known in the art for introducing nucleic acids to cells may be used, such as lipid-mediated carrier transport, chemical-mediated transport, such as calcium phosphate, and the like.
  • the nucleic acid may be introduced along with components that perform one or more of the following activities: enhance nucleic acid uptake by the cell, inhibit annealing of single strands, stabilize the single strands, or other- wise increase inhibition of the target gene.
  • Nucleic acid may be directly introduced into the cell (i.e., intracellularly); or introduced extracellularly into a cavity, interstitial space, into the circulation of an organism, introduced orally or by inhalation, or may be introduced by bathing a cell or organism in a solution containing the nucleic acid.
  • Vascular or extravascular circulation, the blood or lymph system, and the cerebrospinal fluid are sites where the nucleic acid may be introduced.
  • the cell with the target gene may be derived from or contained in any organism.
  • the organism may a plant, animal, protozoan, bacterium, virus, or fungus.
  • the plant may be a monocot, dicot or gymnosperm; the animal may be a vertebrate or invertebrate.
  • Preferred microbes are those used in agriculture or by industry, and those that are pathogenic for plants or animals.
  • sequence identity may be determined by sequence comparison and alignment algorithms known in the art. To determine the percent identity of two nucleic acid sequences (or of two amino acid sequences), the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the first sequence or second sequence for optimal alignment).
  • a preferred, non-limiting example of a local alignment algorithm utilized for the comparison of sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77.
  • siRNA selection Program Greater than 90% sequence identity, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% sequence identity, between the siRNA and the portion of the target gene is preferred.
  • the siRNA may be defined functionally as a nucleotide sequence (or oligonucleotide sequence) that is capable of hybridizing with a portion of the target gene transcript.
  • the oligonucleotide compositions of the present invention can be used to treat any disease involving the expression of a protein.
  • diseases that can be treated by oligonucleotide compositions, just to illustrate, include: cancer, retinopathies, autoimmune diseases, inflammatory diseases (i.e. , ICAM-1 related disorders, Psoriasis, Ulcerative Colitus, Crohn's disease), metabolic, viral diseases (i.e. , HIV, Hepatitis C, flu), miRNA disorders, and cardiovascular diseases.
  • in vitro treatment of cells with oligonucleotides can be used for ex vivo therapy of cells removed from a subject (e.g., for treatment of leukemia or viral infection) or for treatment of cells which did not originate in the subject, but are to be administered to the subject (e.g. , to eliminate transplantation antigen expression on cells to be transplanted into a subject).
  • in vitro treatment of cells can be used in non- therapeutic settings, e.g. , to evaluate gene function, to study gene regulation and protein synthesis or to evaluate improvements made to oligonucleotides designed to modulate gene expression or protein synthesis.
  • In vivo treatment of cells can be useful in certain clinical settings where it is desirable to inhibit the expression of a protein.
  • antisense therapy is reported to be suitable (see, e.g. , U.S. Pat. No. 5,830,653) as well as respiratory syncytial virus infection (WO 95/22,553) influenza virus (WO 94/23,028), and malignancies (WO 94/08,003).
  • respiratory syncytial virus infection WO 95/22,553 influenza virus
  • malignancies WO 94/08,003
  • Other examples of clinical uses of antisense sequences are reviewed, e.g. , in Glaser. 1996. Genetic Engineering News 16: 1.
  • Exemplary targets for cleavage by oligonucleotides include, e.g., protein kinase Ca, ICAM-1, c-raf kinase, p53, c-myb, and the bcr/abl fusion gene found in chronic myelogenous leukemia.
  • the subject nucleic acids can be used in RNAi-based therapy in any animal having
  • RNAi pathway such as human, non-human primate, non-human mammal, non-human vertebrates, rodents (mice, rats, hamsters, rabbits, etc.), domestic livestock animals, pets (cats, dogs, etc.), Xenopus, fish, insects (Drosophila, etc.), and worms (C. elegans), etc.
  • the invention provides methods for inhibiting or preventing in a subject, a disease or condition associated with an aberrant or unwanted target gene expression or activity, by administering to the subject a nucleic acid of the invention. If appropriate, subjects are first treated with a priming agent so as to be more responsive to the subsequent RNAi therapy. Subjects at risk for a disease which is caused or contributed to by aberrant or unwanted target gene expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays known in the art. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the target gene aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending on the type of target gene aberrancy, for example, a target gene, target gene agonist or target gene antagonist agent can be used for treating the subject.
  • the invention pertains to methods of modulating target gene expression, protein expression or activity for therapeutic purposes.
  • the methods of the invention involve contacting a cell capable of expressing target gene with a nucleic acid of the invention that is specific for the target gene or protein (e.g., is specific for the mRNA encoded by said gene or specifying the amino acid sequence of said protein) such that expression or one or more of the activities of target protein is modulated.
  • a nucleic acid of the invention that is specific for the target gene or protein (e.g., is specific for the mRNA encoded by said gene or specifying the amino acid sequence of said protein) such that expression or one or more of the activities of target protein is modulated.
  • the subjects may be first treated with a priming agent so as to be more responsive to the subsequent RNAi therapy if desired.
  • the present invention provides methods of treating a subject afflicted with a disease or disorder characterized by aberrant or unwanted expression or activity of a target gene polypeptide or nucleic acid molecule. Inhibition of target gene activity is desirable in situations in which target gene is abnormally unregulated and/or in which decreased target gene activity is likely to have a beneficial effect.
  • the therapeutic agents of the invention can be administered to subjects to treat (prophylactically or therapeutically) disorders associated with aberrant or unwanted target gene activity.
  • pharmacogenomics i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug
  • Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug.
  • a physician or clinician may consider applying knowledge obtained in relevant pharmacogenomics studies in determining whether to administer a therapeutic agent as well as tailoring the dosage and/or therapeutic regimen of treatment with a therapeutic agent.
  • Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons.
  • the present invention provides compositions and methods to provide polynucleotides in vivo by (1) synthesizing a polynucleotide, which contains hydrophobic entities (2) complexing this hydrophobic modified polynucleotide with a glucan-containing particle, wherein the complex optionally also contains a lipid or amphiphilic molecule; and (3) administering the particle to the patient or animal, for example intravenously, subcutaneously, topically, via catheter locally or orally.
  • Nucleic acid molecules, or compositions comprising nucleic acid molecules, described herein may be used for delivery to any tissue and to inhibit any target gene.
  • the oligonucleotide compositions of the present invention can be used to treat any disease involving the expression of that target gene.
  • the target is MAP4K4.
  • MAP4K4 gene is provided by GenBank Accession number NM_004834.3.
  • the oligonucleotide compositions of the present invention can be used to treat any disease involving the expression of a MAP4K4 protein.
  • the inhibition of a MAP4K4 gene includes any fragments and isoforms of MAP4K4.
  • Diseases that can be treated by oligonucleotide compositions in accordance with the present methods include, just to illustrate: cancer, retinopathies, autoimmune diseases, inflammatory diseases (i.e. , ICAM-1 related disorders, Psoriasis, Ulcerative Colitus, Crohn's disease), viral diseases (i.e. , HIV, Hepatitis C), and cardiovascular diseases.
  • Metabolic disorders or metabolic syndrome, is described by accepted synonyms, which includes, but is not limited to, syndrome X, insulin resistance syndrome, insulin-resistant hypertension, the metabolic hypertensive syndrome, dysmetabolic syndrome.
  • Components of the metabolic syndrome include, but is not limited to, glucose intolerance, impaired glucose tolerance, impaired fasting serum glucose, impaired fasting blood glucose, hyperinsulinemia, pre-diabetes, obesity, visceral obesity, hypertriglyceridemia, elevated serum concentrations of free fatty acids, elevated serum concentrations of C-reactive protein, elevated serum concentrations of lipoprotein(a), elevated serum concentrations of homocysteine, elevated serum concentrations of small, dense low-density lipoprotein (LDL)-cholesterol, elevated serum concentrations of lipoprotein- associated phospholipase (A2), reduced serum concentrations of high density lipoprotein (HDL)-cholesterol, reduced serum concentrations of HDL(2b)-cholesterol, reduced serum concentrations of adiponectin, adipogenesis, and albuminuria.
  • glucose intolerance impaired glucose tolerance
  • impaired fasting serum glucose impaired fasting blood glucose
  • hyperinsulinemia pre-diabetes
  • obesity visceral obesity
  • the target gene is PPIB (peptidylprolyl isomerase B).
  • a representative sequence for the human PPIB gene is provided by GenBank Accession number NM_000942.4.
  • the target gene is SODl (superoxide dismutase 1).
  • a representative sequence for the human SODl gene is provided by GenBank Accession number NM_000454.
  • the target gene is PCSK9 (Proprotein convertase subtilisin kexin 9).
  • a representative sequence for the human PCSK9 gene is provided by GenBank Accession number NM_174936.
  • the oligonucleotide compositions of the present invention can be used to treat any disease involving the expression of a PCSK9 protein.
  • the inhibition of a PCSK9 gene includes any fragments and isoforms of PCSK9.
  • Diseases that can be treated by oligonucleotide compositions in accordance with the present methods include, just to illustrate: metabolic diseases, traits, or conditions in a subject or organism.
  • the metabolic disease is selected from the group consisting of diabetis (e.g., type I and/or type II diabetis), insulin resistance, obesity, or related conditions, including but not limited to sleep apnea, hiatal hernia, reflux esophagisitis, osteoarthritis, gout, cancers associated with weight gain, gallstones, kidney stones, pulmonary hypertension, infertility, cardiovascular disease, above normal weight, and above normal lipid levels, uric acid levels, or oxalate levels.
  • diabetis e.g., type I and/or type II diabetis
  • insulin resistance e.g., type I and/or type II diabetis
  • obesity e.g., type I and/or type II diabetis
  • related conditions including but not limited to sleep apnea, hiatal hernia, reflux esophagisitis, osteoarthritis, gout, cancers associated with weight gain, gallstones, kidney stones, pulmonary hypertension, infertility,
  • PCSK9 diseases that may benefit from the inhibition of PCSK9 include, for example, metabolic disorders, traits and conditions, including but not limited to hyperlipidemia, hypercholesterolemia, cardiovascular disease, atherosclerosis, hypertension, diabetes (e.g., type I and/or type II diabetis), insulin resistance, obesity and/or any other diseases, traits, and conditions that are related to PCSK9 gene expression or activity.
  • metabolic disorders including but not limited to hyperlipidemia, hypercholesterolemia, cardiovascular disease, atherosclerosis, hypertension, diabetes (e.g., type I and/or type II diabetis), insulin resistance, obesity and/or any other diseases, traits, and conditions that are related to PCSK9 gene expression or activity.
  • Metabolic disorders or metabolic syndrome, is described by accepted synonyms, which includes, but is not limited to, syndrome X, insulin resistance syndrome, insulin-resistant hypertension, the metabolic hypertensive syndrome, dysmetabolic syndrome, hyperlipidemia, hypercholesterolemia, cardiovascular disease, atherosclerosis, hypertension, diabetes (e.g., type I and/or type II diabetes), insulin resistance, and obesity.
  • syndrome X insulin resistance syndrome
  • insulin-resistant hypertension the metabolic hypertensive syndrome
  • dysmetabolic syndrome hyperlipidemia
  • hypercholesterolemia hypercholesterolemia
  • cardiovascular disease e.g., atherosclerosis
  • hypertension e.g., type I and/or type II diabetes
  • diabetes e.g., type I and/or type II diabetes
  • obesity e.g., type I and/or type II diabetes
  • Components of the metabolic syndrome include, but is not limited to, glucose intolerance, impaired glucose tolerance, impaired fasting serum glucose, impaired fasting blood glucose, hyperinsulinemia, prediabetes, visceral obesity, hypertriglyceridemia, elevated serum concentrations of free fatty acids, elevated serum concentrations of C-reactive protein, elevated serum concentrations of lipoprotein(a), elevated serum concentrations of homocysteine, elevated serum concentrations of small, dense low-density lipoprotein (LDL)-cholesterol, elevated serum concentrations of lipoprotein-associated phospholipase (A2), reduced serum concentrations of high density lipoprotein (HDL)-cholesterol, reduced serum concentrations of HDL(2b)-cholesterol, reduced serum concentrations of adiponectin, adipogenesis, and albuminuria.
  • the present disclosure may be beneficial and applies to any other diseases, traits, and conditions that are related to PCSK9 gene expression of activity.
  • the invention also encompasses diagnostic uses as well as prophylactic, therapeutic and research uses.
  • Formulations include sterile or non-sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • the invention also includes articles, which refers to any one or collection of components.
  • the articles are kits.
  • the articles include pharmaceutical or diagnostic grade compounds of the invention in one or more containers.
  • the article may include instructions or labels promoting or describing the use of the compounds of the invention.
  • promoted includes all methods of doing business including methods of education, hospital and other clinical instruction, pharmaceutical industry activity including pharmaceutical sales, and any advertising or other promotional activity including written, oral and electronic communication of any form, associated with compositions of the invention in connection with treatment, diagnosis, or prophylaxis of a disease.
  • Instructions can define a component of promotion, and typically involve written instructions on or associated with packaging of compositions of the invention. Instructions also can include any oral or electronic instructions provided in any manner.
  • kits may include one or more containers housing the components of the invention and instructions for use.
  • kits may include one or more agents described herein, along with instructions describing the intended therapeutic or diagnostic application and the proper administration of these agents.
  • agents in a kit may be in a pharmaceutical formulation and dosage suitable for a particular application and for a method of administration of the agents.
  • the kit may be designed to facilitate use of the methods described herein by physicians and can take many forms.
  • Each of the compositions of the kit may be provided in liquid form (e.g., in solution), or in solid form, (e.g., a dry powder).
  • some of the compositions may be constitutable or otherwise processable (e.g., to an active form), for example, by the addition of a suitable solvent or other species (for example, water or a cell culture medium), which may or may not be provided with the kit.
  • a suitable solvent or other species for example, water or a cell culture medium
  • Instructions also can include any oral or electronic instructions provided in any manner such that a user will clearly recognize that the instructions are to be associated with the kit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet, and/or web-based
  • the written instructions may be in a form prescribed by a
  • the kit may contain any one or more of the components described herein in one or more containers.
  • the kit may include instructions for mixing one or more components of the kit and/or isolating and mixing a sample and applying to a subject.
  • the kit may include a container housing agents described herein.
  • the agents may be prepared sterilely, packaged in syringe and shipped refrigerated. Alternatively it may be housed in a vial or other container for storage. A second container may have other agents prepared sterilely.
  • the kit may include the active agents premixed and shipped in a syringe, vial, tube, or other container.
  • the kit may have a variety of forms, such as a blister pouch, a shrink wrapped pouch, a vacuum sealable pouch, a sealable thermoformed tray, or a similar pouch or tray form, with the accessories loosely packed within the pouch, one or more tubes, containers, a box or a bag.
  • the kit may be sterilized after the accessories are added, thereby allowing the individual accessories in the container to be otherwise unwrapped.
  • the kits can be sterilized using any appropriate sterilization techniques, such as radiation sterilization, heat sterilization, or other sterilization methods known in the art.
  • the kit may also include other components, depending on the specific application, for example, containers, cell media, salts, buffers, reagents, syringes, needles, a fabric, such as gauze, for applying or removing a disinfecting agent, disposable gloves, a support for the agents prior to administration etc.
  • other components for example, containers, cell media, salts, buffers, reagents, syringes, needles, a fabric, such as gauze, for applying or removing a disinfecting agent, disposable gloves, a support for the agents prior to administration etc.
  • compositions of the kit may be provided as any suitable form, for example, as liquid solutions or as dried powders.
  • the powder When the composition provided is a dry powder, the powder may be reconstituted by the addition of a suitable solvent, which may also be provided.
  • the liquid form may be concentrated or ready to use.
  • the solvent will depend on the compound and the mode of use or administration. Suitable solvents for drug compositions are well known and are available in the literature. The solvent will depend on the compound and the mode of use or administration.
  • kits in one set of embodiments, may comprise a carrier means being
  • each of the container means comprising one of the separate elements to be used in the method.
  • one of the containers may comprise a positive control for an assay.
  • the kit may include containers for other components, for example, buffers useful in the assay.
  • This article of manufacture includes the appropriate unit dosage form in an appropriate vessel or container such as a glass vial or other container that is hermetically sealed.
  • the active ingredient is sterile and suitable for administration as a particulate free solution.
  • the invention encompasses both parenteral solutions and lyophilized powders, each being sterile, and the latter being suitable for reconstitution prior to injection.
  • the unit dosage form may be a solid suitable for oral, transdermal, topical or mucosal delivery.
  • the unit dosage form is suitable for intravenous, intramuscular or subcutaneous delivery.
  • the invention encompasses solutions, preferably sterile, suitable for each delivery route.
  • ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.
  • a or “an” entity refers to one or more of that entity; for example, “a protein” or “a nucleic acid molecule” refers to one or more of those compounds or at least one compound.
  • a protein or “a nucleic acid molecule” refers to one or more of those compounds or at least one compound.
  • the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably herein. It is also to be noted that the terms “comprising”, “including”, and “having” can be used interchangeably.
  • a compound "selected from the group consisting of refers to one or more of the compounds in the list that follows, including mixtures (i.e., combinations) of two or more of the compounds.
  • an isolated, or biologically pure, protein or nucleic acid molecule is a compound that has been removed from its natural milieu.
  • isolated and biologically pure do not necessarily reflect the extent to which the compound has been purified.
  • An isolated compound of the present invention can be obtained from its natural source, can be produced using molecular biology techniques or can be produced by chemical synthesis.
  • Example 1 Formulation of Neutral Nanotransporters in GeRPs
  • the initial objective was to identify conditions that would allow sif-rxRNA nano /lipid complexes to be trapped in GeRPs.
  • Two glucan particle batches were used: i) 42209 (YGP SAF) and ii) 42209 (YGP+L SAF; this batch theoretically contains natural lipids; ethanol extraction steps were not performed).
  • generation 1 sd-rxRNAs were used (designated herein as duplexes 1 and 2, targeting MM and Map4K4 respectively).
  • Duplex 1 MM control for MAP4K4 sif-rxRNA nano with DY547
  • Duplex 2 Active control for MAP4K4 sif-rxRNA nano with DY547
  • glucan particles were prepared based on protocols described in, and incorporated by reference from, the following references: Soto and Ostroff (2008), "Characterization of multilayered nanoparticles encapsulated in yeast cell wall particles for DNA delivery.” Bioconjug Chem 19(4): 840-8; Soto and Ostroff (2007), “Oral Macrophage Mediated Gene Delivery System,” Nanotech, Volume 2, Chapter 5 (“Drug Delivery”), pages 378-381 ; and Li et al. (2007), "Yeast glucan particles activate murine resident macrophages to secrete proinflammatory cytokines via MyD88-and Syk kinase- dependent pathways.” Clinical Immunology 124(2): 170-181.
  • Duplex 1 MM control for MAP4K4 sif-rxRNA nano with DY547
  • Duplex 2 Active control for MAP4K4 sif-rxRNA nano with DY547
  • the sif-rxRNA nano was used alone in this experiment.
  • Duplex 1 MM control for MAP4K4 sif-rxRNA nano with DY547
  • Duplex 2 Active control for MAP4K4 sif-rxRNA nano with DY547
  • PECs Peritoneal Exudate Cells
  • mice were quickly transferred to a biological safety cabinet, and pins were used to position the mouse ventrally on a surgical board. The animal was washed down with 70% ethanol.
  • Steps 1-5 were repeated with additional mice.
  • Cells from two mice were pooled together in a single 50 ml conical. When cells were harvested from more than two mice, there were multiple tubes of cells.
  • Cell suspensions were filtered over a 70 um cell strainer to remove any small pieces of tissue or fat that may have been in the lavage fluid. The filter was set up over a new sterile 50 ml conical tube. The filter was pre-wet with 1 ml of sterile DPBS (-/-), then cells were poured over the filter. The conical tube that originally contained the cell suspension was rinsed with 5 ml of DPBS (-/-) and then that was added to the filter. 8. Samples were centrifuged at 1500 rpm for 5 min or 1300 rpm for 10 min at room temperature to pellet the cells.
  • Flasks were incubated at 37 °C in a tissue culture incubator at 10 % CO 2 overnight to allow PECs to adhere. *QC step - cells were counted. Samples were spun for 5 min at 1500 rpm and supernatant was removed. Cells were resuspended in 100 ul of Red Blood Cell (RBC) Lysis Buffer and incubated at room temperature for 5 min. Cells were spun for 5 min at 1500 rpm and supernatant was removed. Cells were resuspended in 90 ul of cDMEM and recounted. Day 2
  • RBC Red Blood Cell
  • the 5 ml cell suspension was added to a sterile 50 ml conical tube.
  • Cells were plated in: 6 well plates at a seeding density of lxlO 6 cells/well.
  • Quantigene gene expression assay (version 2.0) was used to detect gene expression as outlined below.
  • the QuantiGene assay can detect the level of gene expression in cell lysates and tissue lysates by utilizing bDNA technology. Generally, samples are incubated overnight in a 96 well plate that has specific probes for the gene of interest. Hybridization of the probes takes place overnight and the next day detection of the hybridization is performed. The signal is amplified and labeled through the use of universal probes provided in the kit. The label probe is enzyme linked so that when a substrate was added to the well, it will give a luminescent signal.
  • the assay was performed as follows:
  • Probe sets were thawed to room temperature (Blocking Reagent, CE, LE, BL) 2. Cell lysates were thawed to room temperature (96 well plate took approximately 20 minutes)
  • Capture plate was allowed to warm to room temperature in foil package for 20-30 minutes.
  • Probe set Master Mix was prepared as follows:
  • Each probe stock was 333.333X.
  • Blocking reagent was 100X.
  • Lysate (example uses 20 ul/well)
  • Wash Buffer was prepared as follows:
  • the plate was removed from the incubator.
  • FIGS 1-3 reveal relative expression of MAPK4K and PPIB following transfection of PECs using duplexes 1 and 2 in NNTs formulated in GeRPs and delivered at 5, 10 or 15 GeRPs/cell.
  • Example 3 Formulation of Neutral Nano transporters in GeRPs using high RNA
  • the next objective was to investigate conditions that would allow sd-rxRNA/lipid complexes to be trapped in GeRPs. Increased loading of GeRPs was investigated. The same two glucan particle batches as described above in Example 1 were used. 1.8 mg of sd-rxRNA per 10 mg batch of glucan particles was tested. For nucleic acid molecules, Map4K4 targeting duplex 4 (generation II; "Gil”) and nontargeting duplex 5 (Gil) were used. As a loading method, 50:50 DOPC:cholesterol in ethanol first, followed by sd-rxRNA was used.
  • Duplex 4 Active control for MAP4K4 sif-rxRNA nano with DY547
  • Duplex 5 MM control for MAP4K4 sif-rxRNA nano with DY547
  • RNAs were pipetted at 37 °C
  • Glucan Particle batch 42209 YGP+L SAF formed a paste; these samples were incubated with the caps closed.
  • the Glucan Particle batch 42209 YGP did not form a paste, they were more like a slurry.
  • These samples were incubated with their caps open to evaporate the ethanol, mixed after 30 minutes, then left to incubate for the remainder 30 minutes. After the 1 hour incubation, most of the excess ethanol had evaporated and the samples were close to a paste.
  • Example 4 Formulation of RNA/peptide complexes in GeRPs using high RNA
  • the next objective was to investigate GeRP loading of rxRNAori or sd-rxRNA and linear or branched peptides.
  • Sd-rxRNA Map4K4 targeting duplex 4 or nontargeting duplex 5 complexed with linear peptide 1 (MW 4230) or branched peptide (MW 11308) to form GeRP cores
  • RNA was added per 10 mg batch of GP (42209 SAF) and trapped with 3.6 mg of peptide (2:1 peptide: RNA)
  • Duplex 6 Active control for PPIB rxRNA ori with DY547
  • Duplex 7 MM control for PPIB rxRNA ori (not labeled)
  • Duplex 4 Active control for MAP4K4 sd-rxRNA with DY547
  • Duplex 5 MM control for MAP4K4 sd-rxRNA with DY547 Methods
  • RNAs were heated to 37 °C before pipetting
  • RNA stocks were concentrated to 44 mg/ml (4 mM)
  • duplex 4 and duplex 5 were at ImM
  • RNA concentration was 1.76 mg
  • Endo-Porter Commercially available, purchased from GeneTools, LLC (the peptide-based reagent Endo-Porter is described in, and incorporated by reference from, US Patent 7,084,248)
  • RNAs used were Duplex 3 (sd-rxRNA) and Duplex 8 (rxRNAori). Each was used separately in the complexes under the conditions described herein.
  • RNA:peptide w/w ratios tested included: 1:1, 1:2.5, 1:5, 1:7.5, 1: 10
  • Endo-Porter was complexed with RNA according to Table 8.
  • RNA/Peptide complexes were diluted 1 :20 and then 5 ul was loaded on the gel (250 ng RNA)
  • RNA/Endo-Porter complexes were diluted 1 :2 and then 5 ul was loaded on the gel (188 ng RNA)
  • MAP4K4 sd-rxRNA GeRPs were found to have a higher transfection efficiency than PPIB rxRNAori GeRPs when fluorescent intensities were compared. Both PPIB rxRNAori GeRPs had extremely low transfection efficiencies based on fluorescent intensity. MAP4K4 sd-rxRNA/MW4230 GeRPs have a slightly higher transfection efficiency than MAP4K4 sd-rxRNA/MW11308 GeRPs when fluorescent intensities are compared.
  • Figures 6 and 7 reveal complex formation between sd-rxRNA nano or rxRNAori with peptides.
  • Figures 8-19 reveal results of transfection of GeRPs containing RNA/peptide complexes in PECs and the corresponding effect on expression of MAPK4K and PPIB. Silencing was observed when GeRPS with duplexes targeting MAP4K4 were transfected. More silencing was observed at higher GeRPs/cell. A dose response was observable between from 5 GeRPs/cell to 20 GeRPs/cell. Observable fluorescence increases with increasing amount of GeRPs/cell. No observable silencing was seen with PPIB targeting GeRPs where minimal fluorescence was observed in pictures, even at 20 GeRPs/cell. No significant toxicity was seen in any condition. Table 9: Oligonucleotides used in Examples 1-4

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Abstract

Des aspects selon l'invention concernent des procédés et des compositions pour l'administration efficace d'acides nucléiques constitués d'acides nucléiques modifiés de nature hydrophobe complexés à des particules de glucane.
PCT/US2011/027165 2010-03-04 2011-03-04 Formulations et procédés d'administration ciblée à des cellules phagocytaires WO2011109698A1 (fr)

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Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8664189B2 (en) 2008-09-22 2014-03-04 Rxi Pharmaceuticals Corporation RNA interference in skin indications
US8815818B2 (en) 2008-07-18 2014-08-26 Rxi Pharmaceuticals Corporation Phagocytic cell delivery of RNAI
WO2014134509A3 (fr) * 2013-02-28 2015-03-12 University Of Massachusetts Particules de glucane modifiées par un peptide et une amine pour la délivrance de charges thérapeutiques
EP2550001A4 (fr) * 2010-03-24 2015-06-03 Rxi Pharmaceuticals Corp Arn interférant dans des indications oculaires
US9074211B2 (en) 2008-11-19 2015-07-07 Rxi Pharmaceuticals Corporation Inhibition of MAP4K4 through RNAI
US9080171B2 (en) 2010-03-24 2015-07-14 RXi Parmaceuticals Corporation Reduced size self-delivering RNAi compounds
US9340786B2 (en) 2010-03-24 2016-05-17 Rxi Pharmaceuticals Corporation RNA interference in dermal and fibrotic indications
US9493774B2 (en) 2009-01-05 2016-11-15 Rxi Pharmaceuticals Corporation Inhibition of PCSK9 through RNAi
US9579394B2 (en) 2011-08-08 2017-02-28 Universitaet Regensburg Polyanion nanocomplexes for therapeutic applications
WO2017044507A3 (fr) * 2015-09-08 2017-05-26 Sirnaomics, Inc. Formulations de nanoparticules/arnsi pour le traitement d'une infection par coronavirus du syndrome respiratoire du moyen-orient
US9745574B2 (en) 2009-02-04 2017-08-29 Rxi Pharmaceuticals Corporation RNA duplexes with single stranded phosphorothioate nucleotide regions for additional functionality
EP3132044A4 (fr) * 2014-04-18 2017-09-13 University of Massachusetts Chargement d'exosomes avec des oligonucléotides hydrophobiquement modifiés
US10131904B2 (en) 2008-02-11 2018-11-20 Rxi Pharmaceuticals Corporation Modified RNAi polynucleotides and uses thereof
WO2019136459A1 (fr) 2018-01-08 2019-07-11 Iovance Biotherapeutics, Inc. Procédés de génération de produits de til enrichis pour des lymphocytes t spécifiques d'un antigène tumoral
WO2019136456A1 (fr) 2018-01-08 2019-07-11 Iovance Biotherapeutics, Inc. Procédés de génération de produits de til enrichis pour des lymphocytes t spécifiques d'un antigène tumoral
US10478503B2 (en) 2016-01-31 2019-11-19 University Of Massachusetts Branched oligonucleotides
US10633653B2 (en) 2015-08-14 2020-04-28 University Of Massachusetts Bioactive conjugates for oligonucleotide delivery
US11702659B2 (en) 2021-06-23 2023-07-18 University Of Massachusetts Optimized anti-FLT1 oligonucleotide compounds for treatment of preeclampsia and other angiogenic disorders
US11753638B2 (en) 2016-08-12 2023-09-12 University Of Massachusetts Conjugated oligonucleotides
US11827882B2 (en) 2018-08-10 2023-11-28 University Of Massachusetts Modified oligonucleotides targeting SNPs
US12024706B2 (en) 2019-08-09 2024-07-02 University Of Massachusetts Modified oligonucleotides targeting SNPs
US12049627B2 (en) 2017-06-23 2024-07-30 University Of Massachusetts Two-tailed self-delivering siRNA
US12077755B2 (en) 2020-03-09 2024-09-03 University Of Massachusetts Bioactive conjugates for oligonucleotide delivery

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040167090A1 (en) * 2003-02-21 2004-08-26 Monahan Sean D. Covalent modification of RNA for in vitro and in vivo delivery
US20050281781A1 (en) * 2004-06-16 2005-12-22 Ostroff Gary R Drug delivery product and methods
US20060014667A1 (en) * 2004-07-14 2006-01-19 Gene Tools, Llc Peptide composition and method for delivering substances into the cytosol of cells
US20090171075A1 (en) * 2007-10-25 2009-07-02 Li Yong-Fu Molecular transporter compositions comprising dendrimeric oligoguanidine with a triazine core that facilitate delivery into cells in vivo
WO2010033247A2 (fr) * 2008-09-22 2010-03-25 Rxi Pharmaceuticals Corporation Composés d'arni de taille réduite à auto-délivrance

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040167090A1 (en) * 2003-02-21 2004-08-26 Monahan Sean D. Covalent modification of RNA for in vitro and in vivo delivery
US20050281781A1 (en) * 2004-06-16 2005-12-22 Ostroff Gary R Drug delivery product and methods
US20060014667A1 (en) * 2004-07-14 2006-01-19 Gene Tools, Llc Peptide composition and method for delivering substances into the cytosol of cells
US20090171075A1 (en) * 2007-10-25 2009-07-02 Li Yong-Fu Molecular transporter compositions comprising dendrimeric oligoguanidine with a triazine core that facilitate delivery into cells in vivo
WO2010033247A2 (fr) * 2008-09-22 2010-03-25 Rxi Pharmaceuticals Corporation Composés d'arni de taille réduite à auto-délivrance

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* Cited by examiner, † Cited by third party
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US10633654B2 (en) 2008-02-11 2020-04-28 Phio Pharmaceuticals Corp. Modified RNAi polynucleotides and uses thereof
US10131904B2 (en) 2008-02-11 2018-11-20 Rxi Pharmaceuticals Corporation Modified RNAi polynucleotides and uses thereof
US8815818B2 (en) 2008-07-18 2014-08-26 Rxi Pharmaceuticals Corporation Phagocytic cell delivery of RNAI
US9175289B2 (en) 2008-09-22 2015-11-03 Rxi Pharmaceuticals Corporation Reduced size self-delivering RNAi compounds
US10876119B2 (en) 2008-09-22 2020-12-29 Phio Pharmaceuticals Corp. Reduced size self-delivering RNAI compounds
US10774330B2 (en) 2008-09-22 2020-09-15 Phio Pharmaceuticals Corp. Reduced size self-delivering RNAI compounds
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US9303259B2 (en) 2008-09-22 2016-04-05 Rxi Pharmaceuticals Corporation RNA interference in skin indications
US8796443B2 (en) 2008-09-22 2014-08-05 Rxi Pharmaceuticals Corporation Reduced size self-delivering RNAi compounds
US8664189B2 (en) 2008-09-22 2014-03-04 Rxi Pharmaceuticals Corporation RNA interference in skin indications
US10041073B2 (en) 2008-09-22 2018-08-07 Rxi Pharmaceuticals Corporation Reduced size self-delivering RNAi compounds
US11396654B2 (en) 2008-09-22 2022-07-26 Phio Pharmaceuticals Corp. Neutral nanotransporters
US9074211B2 (en) 2008-11-19 2015-07-07 Rxi Pharmaceuticals Corporation Inhibition of MAP4K4 through RNAI
US11254940B2 (en) 2008-11-19 2022-02-22 Phio Pharmaceuticals Corp. Inhibition of MAP4K4 through RNAi
US10167471B2 (en) 2009-01-05 2019-01-01 Rxi Pharmaceuticals Corporation Inhibition of PCSK9 through RNAI
US9493774B2 (en) 2009-01-05 2016-11-15 Rxi Pharmaceuticals Corporation Inhibition of PCSK9 through RNAi
US11667915B2 (en) 2009-02-04 2023-06-06 Phio Pharmaceuticals Corp. RNA duplexes with single stranded phosphorothioate nucleotide regions for additional functionality
US9745574B2 (en) 2009-02-04 2017-08-29 Rxi Pharmaceuticals Corporation RNA duplexes with single stranded phosphorothioate nucleotide regions for additional functionality
US10479992B2 (en) 2009-02-04 2019-11-19 Phio Pharmaceuticals Corp. RNA duplexes with single stranded phosphorothioate nucleotide regions for additional functionality
US9095504B2 (en) 2010-03-24 2015-08-04 Rxi Pharmaceuticals Corporation RNA interference in ocular indications
US9963702B2 (en) 2010-03-24 2018-05-08 Rxi Pharmaceuticals Corporation RNA interference in dermal and fibrotic indications
US10184124B2 (en) 2010-03-24 2019-01-22 Phio Pharmaceuticals Corp. RNA interference in ocular indications
US10240149B2 (en) 2010-03-24 2019-03-26 Phio Pharmaceuticals Corp. Reduced size self-delivering RNAi compounds
US11584933B2 (en) 2010-03-24 2023-02-21 Phio Pharmaceuticals Corp. RNA interference in ocular indications
US11118178B2 (en) 2010-03-24 2021-09-14 Phio Pharmaceuticals Corp. Reduced size self-delivering RNAI compounds
US9340786B2 (en) 2010-03-24 2016-05-17 Rxi Pharmaceuticals Corporation RNA interference in dermal and fibrotic indications
US10913948B2 (en) 2010-03-24 2021-02-09 Phio Pharmaceuticals Corp. RNA interference in dermal and fibrotic indications
US9080171B2 (en) 2010-03-24 2015-07-14 RXi Parmaceuticals Corporation Reduced size self-delivering RNAi compounds
EP2550001A4 (fr) * 2010-03-24 2015-06-03 Rxi Pharmaceuticals Corp Arn interférant dans des indications oculaires
US10662430B2 (en) 2010-03-24 2020-05-26 Phio Pharmaceuticals Corp. RNA interference in ocular indications
US9579394B2 (en) 2011-08-08 2017-02-28 Universitaet Regensburg Polyanion nanocomplexes for therapeutic applications
WO2014134509A3 (fr) * 2013-02-28 2015-03-12 University Of Massachusetts Particules de glucane modifiées par un peptide et une amine pour la délivrance de charges thérapeutiques
US11505807B2 (en) 2014-04-18 2022-11-22 University Of Massachusetts Exosomal loading using hydrophobically modified oligonucleotides
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US20230399659A1 (en) * 2014-04-18 2023-12-14 University Of Massachusetts Exosomal loading using hydrophobically modified oligonucleotides
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US10513710B2 (en) 2014-04-18 2019-12-24 University Of Massachusetts Exosomal loading using hydrophobically modified oligonucleotides
EP3132044A4 (fr) * 2014-04-18 2017-09-13 University of Massachusetts Chargement d'exosomes avec des oligonucléotides hydrophobiquement modifiés
US10633653B2 (en) 2015-08-14 2020-04-28 University Of Massachusetts Bioactive conjugates for oligonucleotide delivery
US11058779B2 (en) 2015-09-08 2021-07-13 Sirnaomics, Inc. Sirna/nanoparticle formulations for treatment of middle-east respiratory syndrome coronaviral infection
WO2017044507A3 (fr) * 2015-09-08 2017-05-26 Sirnaomics, Inc. Formulations de nanoparticules/arnsi pour le traitement d'une infection par coronavirus du syndrome respiratoire du moyen-orient
US10478503B2 (en) 2016-01-31 2019-11-19 University Of Massachusetts Branched oligonucleotides
US10799591B2 (en) 2016-01-31 2020-10-13 University Of Massachusetts Branched oligonucleotides
US11896669B2 (en) 2016-01-31 2024-02-13 University Of Massachusetts Branched oligonucleotides
US11753638B2 (en) 2016-08-12 2023-09-12 University Of Massachusetts Conjugated oligonucleotides
US12049627B2 (en) 2017-06-23 2024-07-30 University Of Massachusetts Two-tailed self-delivering siRNA
WO2019136459A1 (fr) 2018-01-08 2019-07-11 Iovance Biotherapeutics, Inc. Procédés de génération de produits de til enrichis pour des lymphocytes t spécifiques d'un antigène tumoral
WO2019136456A1 (fr) 2018-01-08 2019-07-11 Iovance Biotherapeutics, Inc. Procédés de génération de produits de til enrichis pour des lymphocytes t spécifiques d'un antigène tumoral
US11827882B2 (en) 2018-08-10 2023-11-28 University Of Massachusetts Modified oligonucleotides targeting SNPs
US12024706B2 (en) 2019-08-09 2024-07-02 University Of Massachusetts Modified oligonucleotides targeting SNPs
US12077755B2 (en) 2020-03-09 2024-09-03 University Of Massachusetts Bioactive conjugates for oligonucleotide delivery
US11702659B2 (en) 2021-06-23 2023-07-18 University Of Massachusetts Optimized anti-FLT1 oligonucleotide compounds for treatment of preeclampsia and other angiogenic disorders

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