EP1644500A2 - Compositions polycationiques destinees a l'administration cellulaire de polynucleotides - Google Patents

Compositions polycationiques destinees a l'administration cellulaire de polynucleotides

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Publication number
EP1644500A2
EP1644500A2 EP04777833A EP04777833A EP1644500A2 EP 1644500 A2 EP1644500 A2 EP 1644500A2 EP 04777833 A EP04777833 A EP 04777833A EP 04777833 A EP04777833 A EP 04777833A EP 1644500 A2 EP1644500 A2 EP 1644500A2
Authority
EP
European Patent Office
Prior art keywords
substituted
guanidinium
amine
nucleic acid
histidyl
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP04777833A
Other languages
German (de)
English (en)
Inventor
Chandra Vargeese
Wang Weimin
Tongqian Chen
David Sweedler
Peter Haeberli
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sirna Therapeutics Inc
Original Assignee
Sirna Therapeutics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US10/693,059 external-priority patent/US20080039414A1/en
Priority claimed from US10/720,448 external-priority patent/US8273866B2/en
Priority claimed from US10/757,803 external-priority patent/US20050020525A1/en
Priority claimed from US10/826,966 external-priority patent/US20050032733A1/en
Priority claimed from PCT/US2004/016390 external-priority patent/WO2005019453A2/fr
Application filed by Sirna Therapeutics Inc filed Critical Sirna Therapeutics Inc
Publication of EP1644500A2 publication Critical patent/EP1644500A2/fr
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
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    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
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    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
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    • C12Y103/00—Oxidoreductases acting on the CH-CH group of donors (1.3)
    • C12Y103/01—Oxidoreductases acting on the CH-CH group of donors (1.3) with NAD+ or NADP+ as acceptor (1.3.1)
    • C12Y103/01022—3-Oxo-5alpha-steroid 4-dehydrogenase (NADP+) (1.3.1.22), i.e. cortisone alpha-reductase
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    • C12Y604/00—Ligases forming carbon-carbon bonds (6.4)
    • C12Y604/01—Ligases forming carbon-carbon bonds (6.4.1)
    • C12Y604/01002—Acetyl-CoA carboxylase (6.4.1.2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
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    • C12N2310/00—Structure or type of the nucleic acid
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Definitions

  • the present invention relates to the delivery of biologically active molecules to cells.
  • the invention relates to compounds, compositions and methods for delivering nucleic acids, polynucleotides, and oligonucleotides such RNA, DNA and analogs thereof, peptides, polypeptides, proteins, antibodies, hormones and small molecules, to cells by facilitating transport across cellular membranes epithelial tissues and endothelial tissues.
  • the compounds, compositions and methods of the invention are useful in therapeutic, research, and diagnostic applications that rely upon the efficient transfer of biologically active molecules into cells, tissues, and organs.
  • the cellular delivery of various therapeutic compounds is usually compromised by two limitations.
  • Second the trafficking of many compounds into living cells is highly restricted by the complex membrane systems of the cell.
  • Specific transporters allow the selective entry of nutrients or regulatory molecules, while excluding most exogenous molecules such as nucleic acids and proteins.
  • Various strategies can be used to improve transport of compounds into cells, including the use of lipid carriers, biodegradable polymers, and various conjugate systems.
  • Viral vectors can be used to transfer genes efficiently into some cell types, but they generally cannot be used to introduce chemically synthesized molecules into cells.
  • An alternative approach is to use delivery formulations incorporating cationic lipids, which interact with nucleic acids through one end and lipids or membrane systems through another (for a review see Feigner, 1990, Advanced Drug Delivery Reviews, 5,162-187; Feigner 1993, J. Liposome Res., 3,3-16).
  • Synthetic nucleic acids as well as plasmids can be delivered using the cytofectins, although the utility of such compounds is often limited by cell-type specificity, requirement for low serum during transfection, and toxicity.
  • Liposomes are attractive drug carriers since they protect biological molecules from degradation while improving their cellular uptake.
  • polyanions e.g., DNA
  • Lipid aggregates can be formed with macromolecules using cationic lipids alone or including other lipids and amphiphiles such as phosphatidylethanolamine.
  • conjugates are often selected based on the ability of certain molecules to be selectively transported into specific cells, for example via receptor-mediated endocytosis. By attaching a compound of interest to molecules that are actively transported across the cellular membranes, the effective transfer of that compound into cells or specific cellular organelles can be realized. Alternately, molecules that are able to penetrate cellular membranes without active transport mechanisms, for example, various lipophilic molecules, can be used to deliver compounds of interest.
  • molecules that can be utilized as conjugates include but are not limited to peptides, hormones, fatty acids, vitamins, flavonoids, sugars, reporter molecules, reporter enzymes, chelators, porphyrins, intercalcators, and other molecules that are capable of penetrating cellular membranes, either by active transport or passive transport.
  • the delivery of compounds to specific cell types can be accomplished by utilizing receptors associated with specific cell types.
  • Particular receptors are overexpressed in certain cancerous cells, including the high affinity folic acid receptor.
  • the high affinity folate receptor is a tumor marker that is overexpressed in a variety of neoplastic tissues, including breast, ovarian, cervical, colorectal, renal, and nasoparyngeal tumors, but is expressed to a very limited extent in normal tissues.
  • the use of folic acid based conjugates to transport exogenous compounds across cell membranes can provide a targeted delivery approach to the treatment and diagnosis of disease and can provide a reduction in the required dose of therapeutic compounds.
  • bioconjugates including folate bioconjugates.
  • Godwin et al, 1972, J. Biol. Chem., 247, 2266-2271 report the synthesis of biologically active pteroyloligo-L-glutamates.
  • Habus et al, 1998, Bioconjugate Chem., 9, 283-291 describe a method for the solid phase synthesis of certain oligonucleotide-folate conjugates.
  • Cook, US Patent No. 6,721,208 describes certain oligonucleotides modified with specific conjugate groups.
  • biotin and folate conjugates to enhance transmembrane transport of exogenous molecules, including specific oligonucleotides has been reported by Low et al, US Patent Nos. 5,416,016, 5,108,921, and International PCT publication No. WO 90/12096.
  • Manoharan et al, International PCT publication No. WO 99/66063 describe certain folate conjugates, including specific nucleic acid folate conjugates with a phosphoramidite moiety attached to the nucleic acid component of the conjugate, and methods for the synthesis of these folate conjugates. Nomura et al, 2000, J. Org.
  • Chem., 65, 5016-5021 describe the synthesis of an intermediate, alpha-[2- (trimethylsilyl)ethoxycarbonyl]folic acid, useful in the synthesis of ceratin types of folate-nucleoside conjugates.
  • Guzaev et al, US 6,335,434 describes the synthesis of certain folate oligonucleotide conjugates.
  • the delivery of compounds to other cell types can be accomplished by utilizing receptors associated with a certain type of cell, such as hepatocytes.
  • receptors associated with a certain type of cell such as hepatocytes.
  • drug delivery systems utilizing receptor-mediated endocytosis have been employed to achieve drug targeting as well as drug-uptake enhancement.
  • the asialoglycoprotein receptor (ASGPr) see for example Wu and Wu, 1987, J. Biol.
  • Chem. 262, 4429- 4432 is unique to hepatocytes and binds branched galactose-terminal glycoproteins, such as asialoorosomucoid (ASOR). Binding of such glycoproteins or synthetic glycoconjugates to the receptor takes place with an affinity that strongly depends on the degree of branching of the oligosaccharide chain, for example, triatennary structures are bound with greater affinity than biatenarry or monoatennary chains (Baenziger and Fiete, 1980, Cell, 22, 61 1-620; Connolly et al, 1982, J. Biol. Chem., 257, 939-945).
  • ASOR asialoorosomucoid
  • peptide based cellular transporters have been developed by several research groups. These peptides are capable of crossing cellular membranes in vitro and in vivo with high efficiency. Examples of such fusogenic peptides include a 16- amino acid fragment of the homeodomain of ANTENNAPEDIA, a Drosophila transcription factor (Wang et al, 1995, PNAS USA., 92, 3318-3322); a 17-mer fragment representing the hydrophobic region of the signal sequence of Kaposi f ⁇ broblast growth factor with or without NLS domain (Antopolsky et al, 1999, Bioconj.
  • peptides were successfully used as part of an antisense oligodeoxyribonucleotide-peptide conjugate for cell culture transfection without lipids. In a number of cases, such conjugates demonstrated better cell culture efficacy then parent oligonucleotides transfected using lipid delivery. In addition, use of phage display techniques has identified several organ targeting and tumor targeting peptides in vivo (Ruoslahti, 1996, Ann. Rev. Cell Dev. Biol, 12, 697-715).
  • WO 98/52614 describes certain methods and compositions for transporting drugs and macromolecules across biological membranes in which the drug or macromolecule is covalently attached to a transport polymer consisting of from 6 to 25 subunits, at least 50% of which contain a guanidino or amidino side chain.
  • the transport polymers are preferably polyarginine peptides composed of all D-, all L- or mixtures of D- and L-arginine.
  • Rothbard et al, U.S. Patent Application Publication No. 20030082356 describes certain poly-lysine and poly-arginine compounds for the delivery of drugs and other agents across epithelial tissues, including the skin, gastrointestinal tract, pulmonary epithelium and blood brain barcier.
  • the present invention features compounds, compositions, and methods to facilitate delivery of molecules into a biological system, such as cells.
  • the compounds, compositions, and methods provided by the instant invention can impart therapeutic activity by transferring therapeutic compounds across cellular membranes or across one or more layers of epithelial or endothelial tissue.
  • the present invention encompasses the design and synthesis of novel agents for the delivery of molecules, including but not limited to small molecules, lipids, nucleosides, nucleotides, nucleic acids, polynucleotides, oligonucleotides, antibodies, toxins, negatively charged polymers and other polymers, for example proteins, peptides, hormones, carbohydrates, or polyamines, across cellular membranes.
  • Non-limiting examples of polynucleotides that can be delivered across cellular membranes using the compounds and methods of the invention include short interfering nucleic acid (siNA), antisense, enzymatic nucleic acid molecules, 2',5'-oligoadenylate, triplex forming oligonucleotides, aptamers, and decoys.
  • siNA short interfering nucleic acid
  • the transporters described are designed to be used either individually or as part of a multi-component system, with or without degradable linkers.
  • the compounds of the invention generally shown in the Formulae below, when formulated into compositions, are expected to improve delivery of molecules into a number of cell types originating from different tissues, in the presence or absence of serum.
  • the compounds, compositions, and methods of the invention are useful for delivering biologically active molecules (e.g. nucleic acids, polynucleotides, oligonucleotides, peptides, polypeptides, proteins, hormones, antibodies, and small molecules) to cells or across epithelial and endothelial tissues, such as skin, mucous membranes, vasculature tissues, gastrointestinal tissues, blood brain barrier tissues, opthamological tissues, pulmonary tissues, liver tissues, cardiac tissues, kidney tissues etc.).
  • biologically active molecules e.g. nucleic acids, polynucleotides, oligonucleotides, peptides, polypeptides, proteins, hormones, antibodies, and small molecules
  • epithelial and endothelial tissues such as skin, mucous membranes, vasculature tissues, gastrointestinal tissues, blood brain barrier tissues, opthamological tissues, pulmonary tissues, liver tissues, cardiac tissues, kidney tissues etc.
  • the compounds, compositions, and methods of the invention
  • the compounds, compositions, and methods of the invention can increase delivery or availability of biologically active molecules (e.g. nucleic acids, poly nucleotides, oligonucleotides, peptides, polypeptides, proteins, hormones, antibodies, and small molecules) to cells or tissues compared to delivery of the molecules in the absence of the compounds, compositions, and methods of the invention.
  • biologically active molecules e.g. nucleic acids, poly nucleotides, oligonucleotides, peptides, polypeptides, proteins, hormones, antibodies, and small molecules
  • the level of a biologically active molecule inside a cell, tissue, or organism is increased in the presence of the compounds and compositions of the invention compared to when the compounds and compositions ofthe invention are absent.
  • the present invention features a compound having the Formula 1 :
  • X and Y are the same or different and represent an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group, and n represents an integer from 0 to about 20.
  • a compound having Formula 1 is CAS Registry No. 473759-22-7.
  • the present invention features a compound having the Formula 2:
  • X and Y are the same or different and represent an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group, and n represents an integer from about 1 to about 20.
  • the present invention features a compound having the Formula 3 :
  • X and Y are the same or different and represent an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group, and n represents an integer from about 1 to about 20.
  • the present invention features a compound having the Formula 4:
  • X and Y are the same or different and represent an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl groupand each n independently represents an integer from about 1 to about 20.
  • the present invention features a compound having the Formula 5:
  • X and Y are the same or different and represent an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group, and each n independently represents an integer from about 1 to about 20.
  • the present invention features a compound having the Formula 6:
  • X and Y are the same or different and represent an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group, and each n independently represents an integer from 0 to about 20.
  • the present invention features a compound having the Formula 7:
  • X and Y are the same or different and represent an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group
  • R represents H, alkyl, substituted alkyl, aryl, substituted aryl, or a ligand
  • n represents an integer from about 1 to about 20.
  • the present invention features a compound having the Formula 8:
  • X and Y are the same or different and represent an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group
  • R represents H, alkyl, substituted alkyl, aryl, substituted aryl, or a ligand
  • n represents an integer from about 1 to about 20.
  • the present invention features a compound having the Formula 9:
  • X and Y are the same or different and represent an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group
  • R represents H, alkyl, substituted alkyl, aryl, substituted aryl, or a ligand, and each n independently represents an integer from about 1 to about 20.
  • the present invention features a compound having the Formula 10:
  • X and Y are the same or different and represent an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group
  • R represents H, alkyl, substituted alkyl, aryl, substituted aryl, or a ligand
  • each n independently represents an integer from about 1 to about 20.
  • the present invention features a compound having the Formula 11 :
  • X and Y are the same or different and represent an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group that can be the same or different
  • R represents H, alkyl, substituted alkyl, aryl, substituted aryl, or a ligand, and each n independently represents an integer from 0 to about 20.
  • the present invention features a compound having the Formula 12:
  • X and Y are the same or different and represent an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group
  • R represents H, alkyl, substituted alkyl, aryl, substituted aryl, or a ligand
  • each n independently represents an integer from 0 to about 20.
  • the present invention features a compound having the Formula 13 :
  • X and Y are the same or different and represent an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group
  • R represents H, alkyl, substituted alkyl, aryl, substituted aryl, or a ligand, and each n independently represents an integer from 0 to about 20.
  • the present invention features a compound having the Formula 14:
  • X and Y are the same or different and represent an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group
  • R represents H, alkyl, substituted alkyl, aryl, substituted aryl, or a ligand
  • each n independently represents an integer from 0 to about 20, and n' represents an integer from about 1 to about 20.
  • the present invention features a compound having the Formula 15 :
  • X and Y are the same or different and represent an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group
  • R represents H, alkyl, substituted alkyl, aryl, substituted aryl, or a ligand
  • each n independently represents an integer from 0 to about 20, and n' represents an integer from about 1 to about 20.
  • the present invention features a compound having the Formula 16:
  • X and Y are the same or different and represent an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group
  • R represents H, alkyl, substituted alkyl, aryl, substituted aryl, or a ligand
  • each n independently represents an integer from 0 to about 20
  • each n' independently represents an integer from about 1 to about 10.
  • the present invention features a compound having the Formula 17:
  • X and Y are the same or different and represent an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group
  • R represents H, alkyl, substituted alkyl, aryl, substituted aryl, or a ligand, each n independently represents an integer from 0 to about 20, and each n' independently represents an integer from about 1 to about 10.
  • the present invention features a compound having the Formula 18:
  • X and Y are the same or different and represent an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group
  • R represents H, alkyl, substituted alkyl, aryl, substituted aryl, or a ligand
  • each n independently represents an integer from 0 to about 20
  • each n' independently represents an integer from about 1 to about 10.
  • the present invention features a compound having the Formula 19:
  • X, Y and Z are the same or different and represent an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl, and each n independently represents an integer from about 1 to about 20.
  • the present invention features a compound having the Formula 20:
  • X represents an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group
  • n represents an integer from 0 to about 20.
  • the present invention features a compound having the Formula 21 :
  • X represents an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group, and n represents an integer from about 1 to about 20.
  • the present invention features a compound having the Formula 22:
  • X represents an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group, and n represents an integer from about 1 to about 20.
  • the present invention features a compound having the Formula 23 :
  • X represents an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group, and each n independently represents an integer from about 1 to about 20.
  • the present invention features a compound having the Formula 24:
  • X represents an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group, and each n independently represents an integer from about 1 to about 20.
  • the present invention features a compound having the Formula 25:
  • X represents an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group, and each n independently represents an integer from 0 to about 20.
  • the present invention features a compound having the Formula 26:
  • X represents an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group
  • R represents H, alkyl, substituted alkyl, aryl, substituted aryl, or a ligand
  • n represents an integer from about 1 to about 20.
  • the present invention features a compound having the Formula 27:
  • X represents an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group
  • R represents H, alkyl, substituted alkyl, aryl, substituted aryl, or a ligand
  • n represents an integer from about 1 to about 20.
  • the present invention features a compound having the Formula 28:
  • n 28 wherein X represents an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group, R represents H, alkyl, substituted alkyl, aryl, substituted aryl, or a ligand, and each n independently represents an integer from about 1 to about 20.
  • the present invention features a compound having the Formula 29:
  • X represents an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group
  • R represents H, alkyl, substituted alkyl, aryl, substituted aryl, or a ligand
  • each n independently represents an integer from about 1 to about 20.
  • the present invention features a compound having the Formula 30:
  • X represents an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group
  • R represents H, alkyl, substituted alkyl, aryl, substituted aryl, or a ligand, and each n independently represents an integer from 0 to about 20.
  • the present invention features a compound having the Formula 31 :
  • X represents an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group
  • R represents H, alkyl, substituted alkyl, aryl, substituted aryl, or a ligand
  • each n independently represents an integer from 0 to about 20.
  • the present invention features a compound having the Formula 32:
  • X represents an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group
  • R represents H, alkyl, substituted alkyl, aryl, substituted aryl, or a ligand
  • each n independently represents an integer from 0 to about 20.
  • the present invention features a compound having the Formula 33:
  • X represents an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group
  • R represents H, alkyl, substituted alkyl, aryl, substituted aryl, or a ligand
  • each n independently represents an integer from 0 to about 20, and n' represents an integer from about 1 to about 20.
  • the present invention features a compound having the Formula 34:
  • X represents an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group
  • R represents H, alkyl, substituted alkyl, aryl, substituted aryl, or a ligand
  • each n independently represents an integer from 0 to about 20, and n' represents an integer from about 1 to about 20.
  • the present invention features a compound having the Formula 35:
  • X represents an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group
  • R represents H, alkyl, substituted alkyl, aryl, substituted aryl, or a ligand
  • each n independently represents an integer from 0 to about 20
  • each n' independently represents an integer from about 1 to about 10.
  • the present invention features a compound having the Formula 36:
  • X represents an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group
  • R represents H, alkyl, substituted alkyl, aryl, substituted aryl, or a ligand
  • each n independently represents an integer from 0 to about 20
  • each n' independently represents an integer from about 1 to about 10.
  • the present invention features a compound having the Formula 37:
  • X represents an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group
  • R represents H, alkyl, substituted alkyl, aryl, substituted aryl, or a ligand
  • each n independently represents an integer from 0 to about 20
  • each n' independently represents an integer from about 1 to about 10.
  • the present invention features a compound having the Formula 38: z
  • X, and Y are the same or different and represent an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl
  • Z represents a compound having any of Formulae 20-37, and each n independently represents an integer from about 1 to about 20.
  • the present invention features a compound having the Formula 39:
  • X represents an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group
  • L represents a ligand
  • n represents an integer from 0 to about 20.
  • the present invention features a compound having the Formula 40:
  • X represents an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group
  • L represents a ligand
  • n represents an integer from about 1 to about 20.
  • the present invention features a compound having the Formula 41 :
  • X represents an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group
  • L represents a ligand
  • n represents an integer from about 1 to about 20.
  • the present invention features a compound having the Formula 42:
  • X and Y each represent an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group that can be the same or different, and each n independently represents an integer from about 1 to about 20.
  • the present invention features a compound having the Formula 43:
  • X represents an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group
  • L represents a ligand
  • each n independently represents an integer from about 1 to about 20.
  • the present invention features a compound having the Formula 44:
  • X represents an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group
  • L represents a ligand
  • each n independently represents an integer from 0 to about 20.
  • the present invention features a compound having the Formula 45:
  • X represents an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group
  • L represents a ligand
  • R represents H, alkyl, substituted alkyl, aryl, substituted aryl, or a ligand
  • n represents an integer from about 1 to about 20.
  • the present invention features a compound having the Formula 46:
  • X represents an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group
  • L represents a ligand
  • R represents H, alkyl, substituted alkyl, aryl, substituted aryl, or a ligand
  • n represents an integer from about 1 to about 20.
  • the present invention features a compound having the Formula 47:
  • n 47 wherein X represents an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group, L represents a ligand, R represents H, alkyl, substituted alkyl, aryl, substituted aryl, or a ligand, and each n independently represents an integer from about 1 to about 20.
  • the present invention features a compound having the Formula 48:
  • X and Y each represent an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group that can be the same or different
  • R represents H, alkyl, substituted alkyl, aryl, substituted aryl, or a ligand
  • each n independently represents an integer from about 1 to about 20.
  • the present invention features a compound having the Formula 49:
  • X represents an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group
  • L represents a ligand
  • R represents H, alkyl, substituted alkyl, aryl, substituted aryl, or a ligand
  • each n independently represents an integer from 0 to about 20.
  • the present invention features a compound having the Formula 50:
  • X represents an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group
  • L represents a ligand
  • R represents H, alkyl, substituted alkyl, aryl, substituted aryl, or a ligand
  • each n independently represents an integer from 0 to about 20.
  • the present invention features a compound having the Formula 51 :
  • X represents an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group
  • L represents a ligand
  • R represents H, alkyl, substituted alkyl, aryl, substituted aryl, or a ligand
  • each n independently represents an integer from 0 to about 20.
  • the present invention features a compound having the Formula 52:
  • X represents an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group
  • L represents a ligand
  • R represents H, alkyl, substituted alkyl, aryl, substituted aryl, or a ligand
  • each n independently represents an integer from 0 to about 20, and n' represents an integer from about 1 to about 20.
  • the present invention features a compound having the Formula 53:
  • X represents an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group
  • L represents a ligand
  • R represents H, alkyl, substituted alkyl, aryl, substituted aryl, or a ligand
  • each n independently represents an integer from 0 to about 20, and n' represents an integer from about 1 to about 20.
  • the present invention features a compound having the Formula 54:
  • X represents an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group
  • L represents a ligand
  • R represents H, alkyl, substituted alkyl, aryl, substituted aryl, or a ligand
  • each n independently represents an integer from 0 to about 20, and each n' independently represents an integer from about 1 to about 10.
  • the present invention features a compound having the Formula 55:
  • X represents an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group
  • L represents a ligand
  • R represents
  • each n independently represents an integer from 0 to about 20
  • each n' independently represents an integer from about 1 to about 10.
  • the present invention features a compound having the Formula 56:
  • X represents an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group
  • L represents a ligand
  • R represents
  • each n independently represents an integer from 0 to about 20
  • each n' independently represents an integer from about 1 to about 10.
  • the present invention features a compound having the Formula 57:
  • X and Y are the same or different and represent an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group
  • L represents a ligand
  • each n independently represents an integer from about 1 to about 20.
  • the present invention features a compound having the Formula 58:
  • X and Y are the same or different and represent an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group
  • L represents a ligand
  • R represents H, alkyl, substituted alkyl, aryl, substituted aryl, or a ligand
  • each n independently represents an integer from about 1 to about 20, and n' is
  • the present invention features a compound having the Formula 59:
  • X and Y are the same or different and represent an amine, substituted amine, guanidinium, substituted guanidinium, histidyl, or substituted histidyl group
  • L represents a ligand
  • each n independently represents an integer from about 1 to about 20, and n' is 1 or 2.
  • the present invention features a compound having the Formula 60:
  • the L of a compound having any of Formulae 39-57 comprises a ligand, for example a ligand that interacts with a receptor, such as a cell surface receptor, that allows the compound having any of Formulae 39-57 to interact with the receptor.
  • Non-limiting examples of ligands include sugars and carbohydrates such as galactose, galactosamine, and N-acetyl galactosamine; hormones such as estrogen, testosterone, progesterone, glucocortisone, adrenaline, insulin, glucagon, cortisol, vitamin D, thyroid hormone, retinoic acid, and growth hormones; growth factors such as VEGF, EGF, NGF, and PDGF; cholesterol; bile acids; neurotransmitters such as GABA, Glutamate, acetylcholine; NOGO; inostitol triphosphate; diacylglycerol; epinephrine; norepinephrine; Nitric Oxide, peptides, vitamins such as folate and pyridoxine, drugs, antibodies and any other molecule that can interact with a receptor in vivo or in vitro.
  • hormones such as estrogen, testosterone, progesterone, glucocortisone, adrenaline, insulin, gluca
  • the ligand can be attached to a compound of the invention using a linker molecule, such as an amide, amido, carbonyl, ester, peptide, disulphide, silane, nucleoside, abasic nucleoside, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, polyhydrocarbon, phosphate ester, phosphoramidate, thiophosphate, alkylphosphate, or photolabile linker.
  • the linker is a biodegradable linker.
  • the invention features a composition comprising a biologically active molecule complexed with a compound having any of Formula 1-60 or any combination thereof.
  • the invention features a biologically active molecule complexed with a compound having any of Formula 1-60 or any combination thereof.
  • a biologically active molecule of the invention comprises a siNA molecule or a portion thereof.
  • the siNA molecule is chemically modified (see for example Table II).
  • the siNA molecule does not comprise any ribonucleotides.
  • Non-limiting examples of siNA molecules are described in McSwiggen, PCT US04/16390, filed May 24, 2004 McSwiggen, USSN 10/826,966, filed April 16, 2004, McSwiggen et al, USSN 10/444,853, filed May 23, 2003 and Vargeese et al, USSN 10/427,160, filed April 30, 2003, all of which are incorporated by reference herein in their entirety including the drawings.
  • a biologically active molecule of the invention comprises an enzymatic nucleic acid.
  • a biologically active molecule of the invention comprises an antisense nucleic acid, 2-5A nucleic acid chimera, decoy, aptamer, or a portion thereof.
  • a composition of the invention comprises a compound or composition described in Beigelman et al, US Patent No. 6,395,713, and Beigelman et al, USSN 10/036,916, both incorporated by reference herein in their entirety, including the drawings.
  • the invention features a composition, comprising a biologically active molecule independently combined with one or more compounds having any of Formulae 1-60 in a suitable carrier or diluent.
  • the biologically active molecule is a nucleic acid, polynucleotide, oligonucleotide, peptide, polypeptide, protein, hormone, antibody, or small molecule.
  • the biologically active molecule is a siNA molecule or a portion thereof.
  • the invention features a biologically active molecule, for example a siNA molecule, complexed with membrane disruptive agents such as those described in U.S. Patent Application Publication No. 20010007666, incorporated by reference herein in its entirety including the drawings.
  • membrane disruptive agent or agents and the biologically active molecule are also complexed with a cationic lipid or helper lipid molecule, such as those lipids described in U.S. Patent No. 6,235,310, incorporated by reference herein in its entirety including the drawings.
  • the membrane disruptive agent or agents and the biologically active molecule are also complexed with a compound having any of Formulae 1 -60 herein.
  • the invention features a method, comprising combining a biologically active molecule with one or more compounds having any of Formulae 1- 60 under conditions suitable for the biologically active molecule to be complexed with the compound(s) having Formulae 1-60.
  • the biologically active molecule is a nucleic acid, polynucleotide, oligonucleotide, peptide, polypeptide, protein, hormone, antibody, or small molecule.
  • the biologically active molecule is a siNA molecule or a portion thereof.
  • the invention features a method, comprising combining one or more compounds having any of Formulae 1-60 with a biologically active molecule under conditions suitable for the biologically active molecule to be complexed with the compound(s) having Formulae 1-60.
  • the biologically active molecule is a nucleic acid, polynucleotide, oligonucleotide, peptide, polypeptide, protein, hormone, antibody, or small molecule.
  • the biologically active molecule is a siNA molecule or a portion thereof.
  • one or more compounds having any of Formulae 1-60 is adjusted to a pH of about 7 before combining the biologically active molecule.
  • a molar excess e.g.
  • a molar excess e.g. greater than two molar equivalents of the compound(s) having any of Formulae 1-60 is combined with the biologically active molecule such that the biologically active molecule is completely ion paired with the compound(s) having any of Formulae 1-60.
  • a molar excess e.g. greater than two molar equivalents of the biologically active molecule is combined with the compound(s) having any of Formulae 1-60 such that the biologically active molecule is partially ion paired with the compound(s) having any of Formulae 1-60.
  • the invention features a composition comprising a biologically active molecule complexed with a compound of the invention having any of Formulae 1-60 or any combination thereof and a pharmaceutically acceptable carrier or diluent.
  • the invention features a lipoplex comprising a cationic component, a lipid component, and a biologically active molecule component (e.g. siNA).
  • the cationic compounds of the invention e.g. compounds having any of Formulae 1 -60
  • the lipid component can comprise any amphipathic compound as is generally known in the art, or alternately lipid compounds described in U.S. Patent No. 6,235,310 or U.S. Patent No. 6,395,713.
  • the formation of a lipoplex can lead to improved pharmacokinetic properties such as increased half life and increased serum stability of biologically active molecules to be delivered to relevant cells and tissues.
  • the invention features a method of treating a subject, comprising contacting cells of the subject with a composition of the invention under conditions suitable for the treatment. This treatment can comprise the use of one or more other drug therapies under conditions suitable for the treatment.
  • the subject is treated for cancer.
  • Cancer types contemplated by the instant invention include but are not limited to breast cancer, lung cancer, colorectal cancer, brain cancer, esophageal cancer, stomach cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, lymphoma, glioma, or multidrug resistant cancers.
  • the invention features a method of treating a subject infected with a virus, comprising contacting cells of the subject with a pharmaceutical composition of the invention, under conditions suitable for the treatment.
  • This treatment can comprise the use of one or more other drug therapies under conditions suitable for the treatment.
  • the viruses contemplated by the instant invention include but are not limited to H1N, HBV, HCV, CMV, RSV, HSV, poliovirus, influenza, rhinovirus, west nile virus, Ebola virus, foot and mouth virus, papilloma virus, and severe acute respiratory virus (SARS).
  • the invention features a method of treating a subject having a disease or pathologic condition relating to gene expression (i.e. the over- expression or under-expression of a gene, or the expression of a mutant gene), comprising contacting cells of the subject with a pharmaceutical composition of the invention, under conditions suitable for the treatment.
  • This treatment can comprise the use of one or more other drug therapies under conditions suitable for the treatment.
  • the disease or pathologic condition can include cancer, infectious disease, autoimmunity, inflammation, endocrine disorders, muscular dystrophy, renal disease, pulmonary disease, cardiovascular disease, C ⁇ S injury, C ⁇ S disease, neurodegenerative disease such as Alzheimer's disease, Huntington disease, Parkinson's disease, ALS, and epilepsy; birth defects, aging, any other disease or condition related to gene expression.
  • the invention also features methods for generating compounds having Formulae 1-60.
  • the invention features methods for converting an amino group into a guanidinium group. Such methods can be used to generate compounds of the invention bearing guanidinium groups, such as a compound having any of Formulae 1-60, wherein X, Y, or Z is a guanidinium group.
  • the invention features a method (guanidinium method 1) of introducing a guanidinium group to a compound comprising an amino group, such as a compound having any of Formulae 1 -60 wherein any of X, Y, or Z is an amino group, comprising: (a) introducing a protecting group (P) at the primary amine of 1 - H-pyrazole-1-carboxamidine or a salt thereof under conditions suitable to generate a protected 1-H-pyrazole-l-carboxamidine derivative;
  • the invention features a method (guanidinium method 2) of introducing a guanidinium group to a compound comprising an amino group, such as a compound having any of Formulae 1 -60 wherein any of X, Y, or Z is an amino group, comprising: (a) introducing a protecting group (P) at the primary amine and the secondary amine of 1-H-pyrazole-l-carboxamidine or a salt thereof under conditions suitable to generate a bis-protected 1-H-pyrazole-l-carboxamidine derivative;
  • the invention features a method (guanidinium method 3) of introducing a guanidinium group to a compound comprising an amino group, such as a compound having any of Formulae 1-60 wherein any of X, Y, or Z is an amino group, comprising: (a) introducing a protecting group (P) at the primary amine and the secondary amine of 1-H-pyrazole-l-carboxamidine or a salt thereof under conditions suitable to generate a bis-protected 1-H-pyrazole-l-carboxamidine derivative;
  • R-NH2 shown in guanidinium methods 1-3 above comprises a compound having any of Formulae 1-60, wherein any of X, Y, or Z comprises a primary amine.
  • R in guanidinium methods 1-3 above comprises a substituted or unsubstituted straight chain, branched chain, or cyclic alkyl, a polyether, a polyamine, or polyglycol having one or more primary amino groups, such as at either end of a linear compound or at differing ring positions of a cyclic compound
  • P shown in methods 1-3 above comprises an amino protecting group as is known in the art, such as a BOC, t-BOC, CBZ, or Fmoc protecting group.
  • the invention features a method (guanidinium method 4) of introducing a guanidinium group to a compound comprising two amino groups, such as a compound having any of Formulae 1-19 or 38-60, wherein any of X, Y, or Z is an amino group, comprising: (a) introducing a protecting group (P) at the primary amine of 1-H-pyrazole-l-carboxamidine or a salt thereof under conditions suitable to generate a protected 1-H-pyrazole-l-carboxamidine derivative;
  • the invention features a method (guanidinium method 5) of introducing a guanidinium group to a compound comprising two amino groups, such as a compound having any of Formulae 1-19 or 38-60, wherein any of X, Y, or Z is an amino group, comprising: (a) introducing a protecting group (P) at the primary amine and secondary amine of 1-H-pyrazole-l-carboxamidine or a salt thereof under conditions suitable to generate a bis-protected 1-H-pyrazole-l-carboxamidine derivative;
  • the invention features a method (guanidinium method 6) of introducing a guanidinium group to a compound comprising two amino groups, such as a compound having any of Formulae 1-19 or 38-60, wherein any of X, Y, or Z is an amino group, comprising: (a) introducing a protecting group (P) at the primary amine and secondary amine of 1-H-pyrazole-l-carboxamidine or a salt thereof under conditions suitable to generate a bis-protected 1-H-pyrazole-l-carboxamidine derivative;
  • H2N-R-NH2 shown in guanidinium methods 4-6 above comprises a compound having any of Formulae 1-19, 38 or 57, wherein both X and Y and/or Z comprise a primary amine.
  • R in guanidinium methods 4-6 above comprises a substituted or unsubstituted straight chain, branched chain, or cyclic alkyl, a polyether, a polyamine, or polyglycol having one or more primary amino groups, such as at either end of a linear compound or at differing ring positions of a cyclic compound (e.g. para, ortho, or meta substitution of a six membered ring).
  • P shown in guanidinium methods 4-6 above comprises an amino protecting group as is known in the art, such as a BOC, t-BOC, CBZ, or Fmoc protecting group.
  • the formulated compounds and compositions of the invention e.g. complexes of compounds having Formulae 1 -60 and a biologically active molecule
  • the compounds and compositions can be locally administered to relevant tissues ex vivo, or in vivo through injection or infusion pump, with or without their incorporation in biopolymers.
  • the compounds and compositions of the instant invention can be used to treat diseases or conditions known in the art.
  • diseases or conditions known in the art.
  • the patient can be treated, or other appropriate cells can be treated, as is evident to those skilled in the art, individually or in combination with one or more drugs under conditions suitable for the treatment.
  • compositions and biologically active molecules of the invention can be used in combination with other known treatments to treat conditions or diseases.
  • the described molecules can be used in combination with one or more known therapeutic agents to treat breast, lung, prostate, colorectal, brain, esophageal, bladder, pancreatic, cervical, head and neck, and ovarian cancer, melanoma, lymphoma, glioma, multidrug resistant cancers, and or HIV, HBV, HCV, CMV, RSV, HSV, poliovirus, influenza, rhinovirus, west nile virus, Ebola virus, foot and mouth virus, papilloma virus, and SARS virus infection, other cancers and other infectious diseases, autoimmunity, inflammation, endocrine disorders, renal disease, pulmonary disease, cardiovascular disease, CNS injury, CNS disease, neurodegenerative disease, birth defects, aging, any other disease or condition related to gene expression.
  • a series of liposome formulations including one or more compounds of Formulae 1-60 herein that enhance the cellular uptake and transmembrane permeability of biologically active molecules in a variety of cell types.
  • the formulated compounds and compositions of the invention e.g. complexes of compounds having Formulae 1 -60 and a biologically active molecule
  • Another embodiment of the invention encompasses the utility of these compounds for increasing the transport of other impermeable and/or lipophilic compounds into cells.
  • Targeting components include ligands for cell surface receptors including, peptides and proteins, glycolipids, lipids, steroid hormones, second messengers, carbohydrates, and their synthetic variants, for example growth factor, folate, cholesterol, signal peptide, or galactose receptors.
  • the compounds (e.g. compounds having any of Formulae 1-60) of the invention are provided as a surface component of a lipid aggregate, covalently or ionically bound, such as a liposome encapsulated with the predetermined molecule to be delivered.
  • Liposomes which can be unilamellar or multilamellar, can introduce encapsulated material into a cell by different mechanisms.
  • the liposome can directly introduce its encapsulated material into the cell cytoplasm by fusing with the cell membrane.
  • the liposome can be compartmentalized into an acidic vacuole (i.e., an endosome) and its contents released from the liposome and out of the acidic vacuole into the cellular cytoplasm.
  • the invention features a lipid aggregate formulation of the compounds (e.g. compounds having any of Formulae 1-60) and biologically active molecules described herein, including phosphatidylcholine (of varying chain length; e.g., egg yolk phosphatidylcholine), cholesterol, a cationic lipid, and 1 ,2-distearoyl-sn- glycero-3-phosphoethanolamine-polythyleneglycol-2000 (DSPE-PEG2000).
  • the cationic lipid component of this lipid aggregate can be any cationic lipid known in the art such as dioleoyl l,2,-diacyl-3-trimethylammonium-propane (DOTAP).
  • this cationic lipid aggregate comprises a covalently bound compound described in any of the Formulae herein.
  • polyethylene glycol is covalently attached to the compounds (e.g. compounds having any of Formulae 1-60) of the present invention.
  • the attached PEG can be any molecular weight but is preferably between 2000-50,000 daltons.
  • the compounds (e.g. compounds having any of Formulae 1-60) and methods of the present invention are useful for introducing nucleotides, nucleosides, nucleic acid molecules, polynucleotides, oligonucleotides, peptides, polypeptides, proteins, antibodies, lipids, and/or small molecule drugs into a cell.
  • the invention can be used for delivery of therapeutic compounds where the corresponding target site of action exists intracellularly.
  • a compound of the invention is designed to be biodegradable, for example by introducing double bonds to saturated alkyl chains of compounds having any of Formulae 1-60 or by introducing chemical groups or linkers that are biodegradable.
  • biodegradable refers to degradation in a biological system, for example enzymatic degradation or chemical degradation.
  • biologically active molecule refers to compounds or molecules that are capable of eliciting or modifying a biological response in a system.
  • biologically active molecules contemplated by the instant invention include therapeutically active molecules such as antibodies, hormones, antivirals, peptides, proteins, chemotherapeutics, small molecules, vitamins, co- factors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex forming oligonucleotides, 2,5-A chimeras, siNA, dsRNA, allozymes, aptamers, decoys and analogs thereof.
  • Biologically active molecules ofthe invention also include molecules capable of modulating the pharmacokinetics and/or pharmacodynamics of other biologically active molecules, for example lipids and polymers such as polyamines, polyamides, polyethylene glycol and other polyethers.
  • lipids and polymers such as polyamines, polyamides, polyethylene glycol and other polyethers.
  • phospholipid refers to a hydrophobic molecule comprising at least one phosphorus group.
  • a phospholipid can comprise a phosphorus containing group and saturated or unsaturated alkyl group, optionally substituted with OH, COOH, oxo, amine, or substituted or unsubstituted aryl groups.
  • nitrogen containing group refers to any chemical group or moiety comprising a nitrogen or substituted nitrogen.
  • nitrogen containing groups include amines, substituted amines, amides, alkylamines, amino acids such as arginine or lysine, polyamines such as spermine or spermidine, cyclic amines such as pyridines, pyrimidines including uracil, thymine, and cytosine, mo ⁇ holines, phthalimides, and heterocyclic amines such as purines, including guanine and adenine.
  • target molecule refers to nucleic acid molecules, proteins, peptides, antibodies, polysaccharides, lipids, sugars, metals, microbial or cellular metabolites, analytes, pharmaceuticals, and other organic and inorganic molecules that are present in a system.
  • inhibitor or “down-regulate” is meant that the expression of the gene, or level of RNAs or equivalent RNAs encoding one or more protein subunits, or activity of one or more protein subunits, such as pathogenic protein, viral protein or cancer related protein subunit(s), is reduced below that observed in the absence of the compounds or combination of compounds of the invention.
  • inhibition or down-regulation with an siNA molecule preferably is below that level observed in the presence of an inactive or scrambled siNA molecule.
  • inhibition or down-regulation with antisense oligonucleotides is preferably below that level observed in the presence of, for example, an oligonucleotide with scrambled sequence or with mismatches.
  • inhibition or down-regulation of viral or oncogenic RNA, protein, or protein subunits with a compound of the instant invention is greater in the presence of the compound than in its absence.
  • up-regulate is meant that the expression of the gene, or level of RNAs or equivalent RNAs encoding one or more protein subunits, or activity of one or more protein subunits, such as viral or oncogenic protein subunit(s), is greater than that observed in the absence of the compounds or combination of compounds of the invention.
  • the expression of a gene such as a viral or cancer related gene, can be increased in order to treat, prevent, ameliorate, or modulate a pathological condition caused or exacerbated by an absence or low level of gene expression.
  • module is meant that the expression of the gene, or level of RNAs or equivalent RNAs encoding one or more protein subunits, or activity of one or more protein subunit(s) of a protein, for example a viral or cancer related protein is up- regulated or down-regulated, such that the expression, level, or activity is greater than or less than that observed in the absence of the compounds or combination of compounds of the invention.
  • short interfering nucleic acid refers to any nucleic acid molecule capable of inhibiting or down regulating gene expression or viral replication, for example by mediating RNA interference "RNAi” or gene silencing in a sequence-specific manner; see for example Zamore et al, 2000, Cell, 101, 25-33; Bass, 2001 , Nature, 41 1, 428-429; Elbashir et al, 2001, Nature, 411, 494-498; and Kreutzer et al, International PCT Publication No.
  • the siNA can be a double-stranded polynucleotide molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof.
  • the siNA can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self- complementary (i.e.
  • each strand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand; such as where the antisense strand and sense strand form a duplex or double stranded structure, for example wherein the double stranded region is about 15 to about 30, e.g., about 15, 16, 17, 18, 1-9, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 base pairs; the antisense strand comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense strand comprises nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof (e.g., about 15 to about 25 or more nucleotides of the siNA molecule are complementary to the target nucleic acid or a portion thereof).
  • the siNA is assembled from a single oligonucleotide, where the self-complementary sense and antisense regions of the siNA are linked by means of a nucleic acid based or non-nucleic acid-based linker(s).
  • the siNA can be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof.
  • the siNA can be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siNA molecule capable of mediating RNAi.
  • the siNA can also comprise a single stranded polynucleotide having nucleotide sequence complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof (for example, where such siNA molecule does not require the presence within the siNA molecule of nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof), wherein the single stranded polynucleotide can further comprise a terminal phosphate group, such as a 5 '-phosphate (see for example Martinez et al, 2002, Cell, 110, 563-574 and Schwarz et al, 2002, Molecular Cell, 10, 537-568), or 5',3'-diphosphate.
  • a terminal phosphate group such as a 5 '-phosphate (see for example Martinez et al, 2002, Cell, 110, 563-574 and Schwarz et al, 2002, Molecular Cell, 10, 537-568), or 5',3'-diphosphate.
  • the siNA molecule of the invention comprises separate sense and antisense sequences or regions, wherein the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linkers molecules as is known in the art, or are alternately non-covalently linked by ionic interactions, hydrogen bonding, van der waals interactions, hydrophobic interactions, and/or stacking interactions.
  • the siNA molecules ofthe invention comprise nucleotide sequence that is complementary to nucleotide sequence of a target gene.
  • the siNA molecule of the invention interacts with nucleotide sequence of a target gene in a manner that causes inhibition of expression of the target gene.
  • siNA molecules need not be limited to those molecules containing only RNA, but further encompasses chemically-modified nucleotides and non-nucleotides.
  • the short interfering nucleic acid molecules of the invention lack 2'-hydroxy (2'-OH) containing nucleotides.
  • Applicant describes in certain embodiments short interfering nucleic acids that do not require the presence of nucleotides having a 2'-hydroxy group for mediating RNAi and as such, short interfering nucleic acid molecules of the invention optionally do not include any ribonucleotides (e.g., nucleotides having a 2'-OH group).
  • siNA molecules that do not require the presence of ribonucleotides within the siNA molecule to support RNAi can however have an attached linker or linkers or other attached or associated groups, moieties, or chains containing one or more nucleotides with 2'- OH groups.
  • siNA molecules can comprise ribonucleotides at about 5, 10, 20, 30, 40, or 50% of the nucleotide positions.
  • modified short interfering nucleic acid molecules of the invention can also be referred to as short interfering modified oligonucleotides "siMON.”
  • siNA is meant to be equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence specific RNAi, for example short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid, short interfering modified oligonucleotide, chemically-modified siRNA, post- transcriptional gene silencing RNA (ptgsRNA), and others.
  • siRNA short interfering RNA
  • dsRNA double-stranded RNA
  • miRNA micro-RNA
  • shRNA short hairpin RNA
  • ptgsRNA post- transcriptional gene silencing RNA
  • RNAi is meant to be equivalent to other terms used to describe sequence specific RNA interference, such as post transcriptional gene silencing, translational inhibition, or epigenetics.
  • siNA molecules of the invention can be used to epigenetically silence genes at both the post- transcriptional level or the pre-transcriptional level.
  • epigenetic regulation of gene expression by siNA molecules of the invention can result from siNA mediated modification of chromatin structure or methylation pattern to alter gene expression (see, for example, Verdel et al, 2004, Science, 303, 672-676; Pal-Bhadra et al, 2004, Science, 303, 669-672; Allshire, 2002, Science, 291, 1818-1819; Volpe et al, 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al, 2002, Science, 297, 2232-2237).
  • a siNA molecule of the invention is a duplex forming oligonucleotide "DFO", (see, for example, Vaish et al., USSN 10/727,780 filed December 3, 2003 and International PCT Application No. US04/16390, filed May 24, 2004, both of which are hereby incorporated by reference herein in their entireties, including the drawings).
  • a siNA molecule of the invention is a multifunctional siNA, (see for example Jadhav et al, USSN 60/543,480 filed February 10, 2004 and International PCT Application No. US04/16390, filed May 24, 2004, both of which are hereby incorporated by reference herein in their entireties, including the drawings).
  • the multifunctional siNA of the invention can comprise sequence targeting, for example, two regions of RNA.
  • enzymatic nucleic acid molecule refers to a nucleic acid molecule which has complementarity in a substrate binding region to a specified gene target, and also has an enzymatic activity which is active to specifically cleave target RNA. That is, the enzymatic nucleic acid molecule is able to intermolecularly cleave RNA and thereby inactivate a target RNA molecule. These complementary regions allow sufficient hybridization of the enzymatic nucleic acid molecule to the target RNA and thus permit cleavage.
  • nucleic acids can be modified at the base, sugar, and/or phosphate groups.
  • enzymatic nucleic acid is used interchangeably with phrases such as ribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, aptazyme or aptamer-binding ribozyme, regulatable ribozyme, catalytic oligonucleotides, nucleozyme, DNAzyme, RNA enzyme, endoribonuclease, endonuclease, minizyme, leadzyme, oligozyme or DNA enzyme. All of these terminologies describe nucleic acid molecules with enzymatic activity.
  • enzymatic nucleic acid molecules described in the instant application are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target nucleic acid regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart a nucleic acid cleaving and/or ligation activity to the molecule (Cech et al., U.S. Patent No. 4,987,071; Cech et al., 1988, 260 JAMA 3030).
  • asymmetric hairpin as used herein is meant a linear siNA molecule comprising an antisense region, a loop portion that can comprise nucleotides or non-nucleotides, and a sense region that comprises fewer nucleotides than the antisense region to the extent that the sense region has enough complimentary nucleotides to base pair with the antisense region and form a duplex with loop.
  • an asymmetric hairpin siNA molecule of the invention can comprise an antisense region having length sufficient to mediate RNAi in a cell or in vitro system (e.g.
  • the asymmetric hairpin siNA molecule can also comprise a 5 '-terminal phosphate group that can be chemically modified.
  • the loop portion of the asymmetric hairpin siNA molecule can comprise nucleotides, non-nucleotides, linker molecules, or conjugate molecules as described herein.
  • asymmetric duplex as used herein is meant a siNA molecule having two separate strands comprising a sense region and an antisense region, wherein the sense region comprises fewer nucleotides than the antisense region to the extent that the sense region has enough complimentary nucleotides to base pair with the antisense region and form a duplex.
  • an asymmetric duplex siNA molecule of the invention can comprise an antisense region having length sufficient to mediate RNAi in a cell or in vitro system (e.g. about 19 to about 22 nucleotides) and a sense region having about 3 to about 18 nucleotides that are complementary to the antisense region.
  • nucleic acid molecule refers to a molecule having nucleotides.
  • the nucleic acid can be single, double, or multiple stranded and can comprise modified or unmodified nucleotides or non-nucleotides or various mixtures and combinations thereof.
  • enzyme portion or “catalytic domain” as used herein refers to that portion/region of the enzymatic nucleic acid molecule essential for cleavage of a nucleic acid substrate.
  • substrate binding arm or “substrate binding domain” as used herein refers to that portion region of a enzymatic nucleic acid which is able to interact, for example via complementarity (i.e., able to base-pair with), with a portion of its substrate. Preferably, such complementarity is 100%, but can be less if desired.
  • enzymatic nucleic acid of the invention can have binding arms that are contiguous or non-contiguous and can be of varying lengths.
  • the length of the binding arm(s) are preferably greater than or equal to four nucleotides and of sufficient length to stably interact with the target RNA; preferably 12-100 nucleotides; more preferably 14-24 nucleotides long (see for example Werner and Uhlenbeck, supra; Hamman et al., supra; Hampel et al., EP0360257; Berzal-Herrance et al., 1993, EMBO J., 12, 2567-73).
  • the design is such that the length of the binding arms are symmetrical (i.e., each of the binding arms is of the same length; e.g., five and five nucleotides, or six and six nucleotides, or seven and seven nucleotides long) or asymmetrical (i.e., the binding arms are of different length; e.g., six and three nucleotides; three and six nucleotides long; four and five nucleotides long; four and six nucleotides long; four and seven nucleotides long; and the like).
  • sufficient length refers to an oligonucleotide of length great enough to provide the intended function under the expected condition, i.e., greater than or equal to 3 nucleotides.
  • sufficient length means that the binding arm sequence is long enough to provide stable binding to a target site under the expected binding conditions. Preferably, the binding arms are not so long as to prevent useful turnover of the nucleic acid molecule.
  • the length of a siNA molecule is of length sufficient to mediate RNAi activity.
  • stably interact refers to interaction of the oligonucleotides with target nucleic acid (e.g., by forming hydrogen bonds with complementary nucleotides in the target under physiological conditions) that is sufficient to the intended purpose (e.g., mediated of RNAi or cleavage of target RNA by an enzyme).
  • antisense nucleic acid refers to a non-enzymatic nucleic acid molecule that binds to target RNA by means of RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al., 1993 Nature 365, 566) interactions and alters the activity of the target RNA (for a review, see Stein and Cheng, 1993 Science 261, 1004 and Woolf et al., US patent No. 5,849,902).
  • antisense molecules are complementary to a target sequence along a single contiguous sequence of the antisense molecule.
  • an antisense molecule can bind to substrate such that the substrate molecule forms a loop, and/or an antisense molecule can bind such that the antisense molecule forms a loop.
  • the antisense molecule can be complementary to two (or even more) non-contiguous substrate sequences or two (or even more) non-contiguous sequence portions of an antisense molecule can be complementary to a target sequence or both.
  • antisense DNA can be used to target RNA by means of DNA-RNA interactions, thereby activating RNase H, which digests the target RNA in the duplex.
  • the antisense oligonucleotides can comprise one or more RNAse H activating region, which is capable of activating RNAse H cleavage of a target RNA.
  • Antisense DNA can be synthesized chemically or expressed via the use of a single stranded DNA expression vector or equivalent thereof.
  • RNase H activating region refers to a region (generally greater than or equal to 4-25 nucleotides in length, preferably from 5-11 nucleotides in length) of a nucleic acid molecule capable of binding to a target RNA to form a non-covalent complex that is recognized by cellular RNase H enzyme (see for example Arrow et al., US 5,849,902; Arrow et al., US 5,989,912).
  • the RNase H enzyme binds to the nucleic acid molecule-target RNA complex and cleaves the target RNA sequence.
  • the RNase H activating region comprises, for example, phosphodiester, phosphorothioate (preferably at least four of the nucleotides are phosphorothiote substitutions; more specifically, 4-1 1 of the nucleotides are phosphorothiote substitutions); phosphorodithioate, 5'-thiophosphate, or methylphosphonate backbone chemistry or a combination thereof.
  • the RNase H activating region can also comprise a variety of sugar chemistries.
  • the RNase H activating region can comprise deoxyribose, arabino, fluoroarabino or a combination thereof, nucleotide sugar chemistry.
  • 2-5A antisense chimera refers to an antisense oligonucleotide containing a 5 '-phosphorylated 2'-5'-linked adenylate residue. These chimeras bind to target RNA in a sequence-specific manner and activate a cellular 2- 5A-dependent ribonuclease which, in turn, cleaves the target RNA (Torrence et al., 1993 Proc. Natl. Acad. Sci. USA 90, 1300; Silverman et al., 2000, Methods Enzymol., 313, 522-533; Player and Torrence, 1998, Pharmacol. Ther., 78, 55-1 13).
  • triplex forming oligonucleotides refers to an oligonucleotide that can bind to a double-stranded DNA in a sequence-specific manner to form a triple-strand helix. Formation of such triple helix structure has been shown to inhibit transcription of the targeted gene (Duval-Valentin et al., 1992 Proc. Natl. Acad. Sci. USA 89, 504; Fox, 2000, Curr. Med. Chem., 7, 17-37; Praseuth et. al., 2000, Biochim. Biophys. Acta, 1489, 181-206).
  • RNA refers to a nucleic acid that encodes an RNA, for example, nucleic acid sequences including but not limited to structural genes encoding a polypeptide.
  • pathogenic protein refers to endogenous or exogenous proteins that are associated with a disease state or condition, for example a particular cancer or viral infection.
  • complementarity refers to the ability of a nucleic acid to form hydrogen bond(s) with another RNA sequence by either traditional Watson-Crick or other non-traditional types.
  • the binding free energy for a nucleic acid molecule with its target or complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., RNA interference, enzymatic nucleic acid cleavage, antisense or triple helix inhibition. Determination of binding free energies for nucleic acid molecules is well known in the art (see, e.g., Turner et al., 1987, CSH Symp. Quant. Biol.
  • a percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary).
  • RNA is meant a molecule comprising at least one ribonucleotide residue.
  • ribonucleotide is meant a nucleotide with a hydroxyl group at the 2' position of a ⁇ -D-ribo-furanose moiety.
  • RNA double-stranded RNA
  • single-stranded RNA isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides.
  • alterations can include addition of non-nucleotide material, such as to the end(s) of the siNA or internally, for example at one or more nucleotides of the RNA.
  • Nucleotides in the RNA molecules of the instant invention can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleo tides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.
  • ribonucleotide or "2'-OH” is meant a nucleotide with a hydroxyl group at the 2' position of a ⁇ -D-ribo-furanose moiety.
  • decoy RNA refers to a RNA molecule or aptamer that is designed to preferentially bind to a predetermined ligand. Such binding can result in the inhibition or activation of a target molecule.
  • the decoy RNA or aptamer can compete with a naturally occurring binding target for the binding of a specific ligand. For example, it has been shown that over-expression of HIV trans-activation response (TAR) RNA can act as a "decoy” and efficiently binds HIV tat protein, thereby preventing it from binding to TAR sequences encoded in the HIV RNA (Sullenger et al., 1990, Cell, 63, 601-608).
  • TAR HIV trans-activation response
  • a decoy RNA can be designed to bind to a receptor and block the binding of an effector molecule or a decoy RNA can be designed to bind to receptor of interest and prevent interaction with the receptor.
  • ssRNA single stranded RNA
  • mRNA messenger RNA
  • tRNA transfer RNA
  • rRNA ribosomal RNA
  • ssDNA single stranded DNA
  • ssDNA single stranded DNA
  • a ssDNA can be a sense or antisense gene sequence or EST (Expressed Sequence Tag).
  • double stranded RNA or “dsRNA” as used herein refers to a double stranded RNA molecule capable of RNA interference, including short interfering RNA (siNA).
  • siNA short interfering RNA
  • allozyme refers to an allosteric enzymatic nucleic acid molecule, see for example see for example George et al., US Patent Nos. 5,834,186 and 5,741,679, Shih et al, US Patent No. 5,589,332, Nathan et al affect US Patent No 5,871,914, Nathan and Ellington, International PCT publication No. WO 00/24931, Breaker et al. International PCT Publication Nos. WO 00/26226 and 98/27104, and Sullenger et al. International PCT publication No. WO 99/29842.
  • the cell can, for example, be in vitro, e.g., in cell culture, or present in a multicellular organism, including,, e.g., birds, plants and mammals such as humans, cows, sheep, apes, monkeys, swine, dogs, and cats.
  • the cell can be prokaryotic (e.g., bacterial cell) or eukaryotic (e.g., mammalian or plant cell).
  • highly conserved sequence region refers to a nucleotide sequence of one or more regions in a target gene does not vary significantly from one generation to the other or from one biological system to the other.
  • non-nucleotide refers to any group or compound which can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including either sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their enzymatic activity.
  • the group or compound is abasic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil or thymine.
  • nucleotide refers to a heterocyclic nitrogenous base in N-glycosidic linkage with a phosphorylated sugar. Nucleotides are recognized in the art to include natural bases (standard), and modified bases well known in the art. Such bases are generally located at the 1' position of a nucleotide sugar moiety. Nucleotides generally comprise a base, sugar and a phosphate group.
  • the nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and other; see for example, Usman and McSwiggen, supra; Eckstein et al, International PCT Publication No. WO 92/07065; Usman et al, International PCT Publication No. WO 93/15187; Uhlman & Peyman, supra all are hereby incorporated by reference herein).
  • modified nucleic acid bases known in the art as summarized by Limbach et al, 1994, Nucleic Acids Res. 22, 2183.
  • nucleic acids include, for example, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6- alkylpyrimidines (e.g. 6-methyluridine), propyne, quesosine, 2-thiouridine, 4- thiouridine, wybutosine, wybutoxosine, 4-acetylcytidine, 5-
  • modified bases in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at 1' position or their equivalents; such bases can be used at any position, for example, within the catalytic core of an enzymatic nucleic acid molecule and/or in the substrate-binding regions of the nucleic acid molecule.
  • nucleoside refers to a heterocyclic nitrogenous base in N-glycosidic linkage with a sugar. Nucleosides are recognized in the art to include natural bases (standard), and modified bases well known in the art. Such bases are generally located at the 1' position of a nucleoside sugar moiety. Nucleosides generally comprise a base and sugar group.
  • the nucleosides can be unmodified or modified at the sugar, and/or base moiety, (also referred to interchangeably as nucleoside analogs, modified nucleosides, non-natural nucleosides, non-standard nucleosides and other; see for example, Usman and McSwiggen, supra; Eckstein et al, International PCT Publication No. WO 92/07065; Usman et al, International PCT Publication No. WO 93/15187; Uhlman & Peyman, supra all are hereby incorporated by reference herein).
  • modified nucleic acid bases known in the art as summarized by Limbach et al, 1994, Nucleic Acids Res. 22, 2183.
  • nucleic acids Some of the non-limiting examples of chemically modified and other natural nucleic acid bases that can be introduced into nucleic acids include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3- methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g.
  • 6-methyluridine 6-methyluridine
  • 2-thiouridine 4-thiouridine
  • wybutosine wybutoxosine
  • 4- acetylcytidine 5-(carboxyhydroxymethyl)uridine, 5 '-carboxymethylaminomethyl-2- thiouridine, 5-carboxymethylaminomethyluridine, beta-D-galactosylqueosine, 1- methyladenosine, 1 -methylinosine, 2,2-dimethylguanosine, 3-methylcytidine
  • 5- methoxyaminomethyl-2-thiouridine 5-methylaminomethyluridine, 5- methylcarbonylmethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 2- methylthio-N6-isopentenyladenosine, beta-D-mannosylqueo
  • modified bases in this aspect is meant nucleoside bases other than adenine, guanine, cytosine and uracil at 1 ' position or their equivalents; such bases can be used at any position, for example, within the catalytic core of an enzymatic nucleic acid molecule and/or in the substrate- binding regions of the nucleic acid molecule.
  • cap structure refers to chemical modifications, which have been incorporated at either terminus of the oligonucleotide (see for example Wincott et al, WO 97/26270, incorporated by reference herein). These terminal modifications protect the nucleic acid molecule from exonuclease degradation, and can help in delivery and/or localization within a cell.
  • the cap can be present at the 5 '-terminus (5 '-cap) or at the 3 '-terminus (3 '-cap) or can be present on both terminus.
  • the 5 '-cap includes inverted abasic residue (moiety), 4',5'-methylene nucleotide; 1 -(beta-D-erythrofiiranosyl) nucleotide, 4'-thio nucleotide, carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; tbreo- pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3 '-3 '-inverted nucleotide moiety; 3 '-3 '-inverted abasic moiety; 3'-2'-inverted nu
  • abasic refers to sugar moieties lacking a base or having other chemical groups in place of a base at the 1' position, for example a 3',3'- linked or 5 ',5 '-linked deoxyabasic ribose derivative (for more details see Wincott et al, International PCT publication No. WO 97/26270).
  • unmodified nucleoside refers to one of the bases adenine, cytosine, guanine, thymine, uracil joined to the 1' carbon of ⁇ -D-ribo- furanose.
  • modified nucleoside refers to any nucleotide base which contains a modification in the chemical structure of an unmodified nucleotide base, sugar and/or phosphate.
  • subject is meant an organism, which is a donor or recipient of explanted cells or the cells themselves. “Subject” also refers to an organism to which the nucleic acid molecules of the invention can be administered.
  • a subject can be a mammal or mammalian cells, including a human or human cells.
  • enhanced enzymatic activity includes activity measured in cells and/or in vivo where the activity is a reflection of both the catalytic activity and the stability of the nucleic acid molecules of the invention.
  • the product of these properties can be increased in vivo compared to an all RNA enzymatic nucleic acid or all DNA enzyme.
  • the activity or stability of the nucleic acid molecule can be decreased (i.e., less than ten-fold), but the overall activity ofthe nucleic acid molecule is enhanced, in vivo.
  • negatively charged molecules refers to molecules such as nucleic acid molecules (e.g., RNA, DNA, oligonucleotides, mixed polymers, peptide nucleic acid, and the like), peptides (e.g., polyaminoacids, polypeptides, proteins and the like), nucleotides, pharmaceutical and biological compositions, that have negatively charged groups that can ion-pair with the positively charged head group of the cationic lipids of the invention.
  • nucleic acid molecules e.g., RNA, DNA, oligonucleotides, mixed polymers, peptide nucleic acid, and the like
  • peptides e.g., polyaminoacids, polypeptides, proteins and the like
  • nucleotides e.g., pharmaceutical and biological compositions, that have negatively charged groups that can ion-pair with the positively charged head group of the cationic lipids of the invention.
  • Coupled refers to a reaction, either chemical or enzymatic, in which one atom, moiety, group, compound or molecule is joined to another atom, moiety, group, compound or molecule.
  • deprotection or “deprotecting” as used herein, refers to the removal of a protecting group.
  • guanidinium refers to a chemical group having the general formula: including any salts thereof and where R is H, or wherein the term “substituted guandinium” is employed, R is alkyl or substituted alkyl.
  • R including any salts thereof and where R is H, or wherein the term "substituted histidyl" is employed, R is alkyl or substituted alkyl.
  • alkyl refers to a saturated aliphatic hydrocarbon, including straight-chain, branched-chain “isoalkyl", and cyclic alkyl groups.
  • alkyl also comprises alkoxy, alkyl-thio, alkyl-thio-alkyl, alkoxyalkyl, alkylamino, alkenyl, alkynyl, alkoxy, cycloalkenyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl, C1-C6 hydrocarbyl, aryl or substituted aryl groups.
  • the alkyl group can comprise 1 to 12 carbons.
  • the alkyl is a lower alkyl of from about 1 to about 7 carbons, or about 1 to about 4 carbons.
  • the alkyl group can be substituted or unsubstituted.
  • the substituted group(s) can comprise hydroxy, oxy, thio, amino, nitro, cyano, alkoxy, alkyl-thio, alkyl-thio-alkyl, alkoxyalkyl, alkylamino, silyl, alkenyl, alkynyl, alkoxy, cycloalkenyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl, C1-C6 hydrocarbyl, aryl or substituted aryl groups.
  • alkyl also includes alkenyl groups containing at least one carbon-carbon double bond, including straight-chain, branched-chain, and cyclic groups.
  • the alkenyl group has about 2 to about 12 carbons.
  • the alkenyl is a lower alkenyl of from about 2 to about 7 carbons, or about 2 to about 4 carbons.
  • the alkenyl group can be substituted or unsubstituted.
  • the substituted group(s) can comprise hydroxy, oxy, thio, amino, nitro, cyano, alkoxy, alkyl-thio, alkyl-thio-alkyl, alkoxyalkyl, alkylamino, silyl, alkenyl, alkynyl, alkoxy, cycloalkenyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl, C1-C6 hydrocarbyl, aryl or substituted aryl groups.
  • alkyl also includes alkynyl groups containing at least one carbon-carbon triple bond, including straight-chain, branched-chain, and cyclic groups.
  • the alkynyl group has about 2 to about 12 carbons. In another embodiment, the alkynyl is a lower alkynyl of from about 2 to about 7 carbons, or about 2 to about 4 carbons. The alkynyl group can be substituted or unsubstituted.
  • the substituted group(s) can comprise hydroxy, oxy, thio, amino, nitro, cyano, alkoxy, alkyl-thio, alkyl-thio-alkyl, alkoxyalkyl, alkylamino, silyl, alkenyl, alkynyl, alkoxy, cycloalkenyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl, C1-C6 hydrocarbyl, aryl or substituted aryl groups.
  • Alkyl groups or moieties of the invention can also include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups.
  • aryl groups can comprise halogen, trihalomethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups.
  • An "alkylaryl” group refers to an alkyl group (as described above) covalently joined to an aryl group (as described above).
  • Carbocyclic aryl groups are groups wherein the ring atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted.
  • Heterocyclic aryl groups are groups having from about 1 to about 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms.
  • Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted.
  • An "amide” refers to an - C(0)-NH-R, where R is either alkyl, aryl, alkylaryl or hydrogen.
  • An “ester” refers to an -C(0)-OR', where R is either alkyl, aryl, alkylaryl or hydrogen.
  • alkoxyalkyl refers to an alkyl-O-alkyl ether, for example, methoxyethyl or ethoxymethyl.
  • alkyl-thio-alkyl refers to an alkyl-S-alkyl thioether, for example, methylthiomethyl or methylthioethyl.
  • amino refers to a nitrogen containing group as is known in the art derived from ammonia by the replacement of one or more hydrogen radicals by organic radicals.
  • aminoacyl and “aminoalkyl” refer to specific N-substituted organic radicals with acyl and alkyl substituent groups respectively.
  • amine is meant a radical with the general formula -NHR or -NR 2 , wherein each R is independently hydrogen, or wherein the term “substituted amine” is employed, R is alkyl or substituted alkyl.
  • animation as used herein refers to a process in which an amino group or substituted amine is introduced into an organic molecule.
  • complex refers to a mixture of one or more compounds that are associated via covalent or non-covalent interactions such as electrostatic interactions, hydrogen bonding interactions, and hydrophobic interactions.
  • complexed refers to the process of combining one or more compounds to generate a complex.
  • a complexed formulation or composition of the invention can be designed to have a net positive charge, a net negative charge, or a neutral charge depending on the ratio of differing compounds used to generate the complex.
  • a compound having any for Formulae 1-60 can be combined with a biologically active molecule and optionally another molecule, such as a lipid, to form a complex in a form or manner suitable for administration to a cell or subject.
  • exocyclic amine protecting moiety refers to a nucleobase amino protecting group compatible with oligonucleotide synthesis, for example, an acyl or amide group.
  • alkenyl refers to a straight or branched hydrocarbon of a designed number of carbon atoms containing at least one carbon-carbon double bond. Examples of “alkenyl” include vinyl, allyl, and 2-methyl-3-heptene.
  • alkoxy refers to an alkyl group of indicated number of carbon atoms attached to the parent molecular moiety through an oxygen bridge.
  • alkoxy groups include, for example, methoxy, ethoxy, propoxy and isopropoxy.
  • alkynyl refers to a straight or branched hydrocarbon of a designed number of carbon atoms containing at least one carbon-carbon triple bond.
  • alkynyl include propargyl, propyne, and 3-hexyne.
  • aryl refers to an aromatic hydrocarbon ring system containing at least one aromatic ring. The aromatic ring can optionally be fused or otherwise attached to other aromatic hydrocarbon rings or non-aromatic hydrocarbon rings.
  • aryl groups include, for example, phenyl, naphthyl, 1,2,3,4- tetrahydronaphthalene and biphenyl.
  • Preferred examples of aryl groups include phenyl and naphthyl.
  • cycloalkenyl refers to a C3-C8 cyclic hydrocarbon containing at least one carbon-carbon double bond.
  • examples of cycloalkenyl include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadiene, cyclohexenyl, 1,3- cyclohexadiene, cycloheptenyl, cycloheptatrienyl, and cyclooctenyl.
  • cycloalkyl refers to a C3-C8 cyclic hydrocarbon.
  • examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.
  • cycloalkylalkyl refers to a C3-C7 cycloalkyl group attached to the parent molecular moiety through an alkyl group, as defined above.
  • alkyl group as defined above.
  • examples of cycloalkylalkyl groups include cyclopropylmethyl and cyclopentylethyl.
  • halogen or halo as used herein refers to indicate fluorine, chlorine, bromine, and iodine.
  • heterocycloalkyl refers to a non-aromatic ring system containing at least one heteroatom selected from nitrogen, oxygen, and sulfur.
  • the heterocycloalkyl ring can be optionally fused to or otherwise attached to other heterocycloalkyl rings and/or non-aromatic hydrocarbon rings.
  • Preferred heterocycloalkyl groups have from 3 to 7 members. Examples of heterocycloalkyl groups include, for example, piperazine, morpholine, piperidine, tetrahydrofuran, pyrrolidine, and pyrazole.
  • Preferred heterocycloalkyl groups include piperidinyl, piperazinyl, morpholinyl, and pyrolidinyl.
  • heteroaryl refers to an aromatic ring system containing at least one heteroatom selected from nitrogen, oxygen, and sulfur.
  • the heteroaryl ring can be fused or otherwise attached to one or more heteroaryl rings, aromatic or non-aromatic hydrocarbon rings or heterocycloalkyl rings.
  • heteroaryl groups include, for example, pyridine, furan, thiophene, 5,6,7,8- tetrahydroisoquinoline and pyrimidine.
  • heteroaryl groups include thienyl, benzothienyl, pyridyl, quinolyl, pyrazinyl, pyrimidyl, imidazolyl, benzimidazolyl, furanyl, benzofuranyl, thiazolyl, benzothiazolyl, isoxazolyl, oxadiazolyl, isothiazolyl, benzisothiazolyl, triazolyl, tetrazolyl, pyrrolyl, indolyl, pyrazolyl, and benzopyrazolyl.
  • C1-C6 hydrocarbyl refers to straight, branched, or cyclic alkyl groups having 1-6 carbon atoms, optionally containing one or more carbon-carbon double or triple bonds.
  • hydrocarbyl groups include, for example, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, 2- pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl, 3-methylpentyl, vinyl, 2-pentene, cyclopropylmethyl, cyclopropyl, cyclohexylmethyl, cyclohexyl and propargyl.
  • C1-C6 hydrocarbyl containing one or two double or triple bonds it is understood that at least two carbons are present in the alkyl for one double or triple bond, and at least four
  • protecting group refers to groups known in the art that are readily introduced and removed from an atom, for example O, N, P, or S. Protecting groups are used to prevent undesirable reactions from taking place that can compete with the formation of a specific compound or intermediate of interest. See also “Protective Groups in Organic Synthesis", 3rd Ed, 1999, Greene, T. W. and related publications.
  • nitrogen protecting group refers to groups known in the art that are readily introduced on to and removed from a nitrogen. Examples of nitrogen protecting groups include Boc, Cbz, benzoyl, and benzyl. See also
  • hydroxy protecting group refers to groups known in the art that are readily introduced on to and removed from an oxygen, specifically an -OH group.
  • hyroxy protecting groups include trityl or substituted trityl goups, such as monomethoxytrityl and dimethoxytrityl, or substituted silyl groups, such as tert-butyldimethyl, trimethylsilyl, or tert- butyldiphenyl silyl groups. See also “Protective Groups in Organic Synthesis", 3rd Ed, 1999, Greene, T. W. and related publications.
  • acyl as used herein refers to -C(0)R groups, wherein R is an alkyl or aryl.
  • phosphorus containing group refers to a chemical group containing a phosphorus atom.
  • the phosphorus atom can be trivalent or pentavalent, and can be substituted with O, H, N, S, C or halogen atoms.
  • Examples of phosphorus containing groups of the instant invention include but are not limited to phosphorus atoms substituted with O, H, N, S, C or halogen atoms, comprising phosphonate, alkylphosphonate, phosphate, diphosphate, triphosphate, pyrophosphate, phosphorothioate, phosphorodithioate, phosphoramidate, phosphoramidite groups, nucleotides and nucleic acid molecules.
  • ligand refers to any compound or molecule, such as a drug, peptide, hormone, or neurotransmitter, that is capable of interacting with another compound, such as a receptor, either directly or indirectly.
  • the receptor that interacts with a ligand can be present on the surface of a cell or can alternately be an intercellular receptor. Interaction of the ligand with the receptor can result in a biochemical reaction, or can simply be a physical interaction or association.
  • Non-limiting examples of ligands include sugars and carbohydrates such as galactose, galactosamine, and N-acetyl galactosamine; hormones such as estrogen, testosterone, progesterone, glucocortisone, adrenaline, insulin, glucagon, cortisol, vitamin D, thyroid hormone, retinoic acid, and growth hormones; growth factors such as VEGF, EGF, NGF, and PDGF; cholesterol; bile acids; neurotransmitters such as GABA, Glutamate, acetylcholine; NOGO; inostitol triphosphate; diacylglycerol; epinephrine; norepinephrine; Nitric Oxide, peptides, vitamins such as folate and pyridoxine, drugs, antibodies and any other molecule that can interact with a receptor in vivo or in vitro.
  • hormones such as estrogen, testosterone, progesterone, glucocortisone, adrenaline, insulin, gluca
  • the ligand can be attached to a compound of the invention using a linker molecule, such as an amide, amido, carbonyl, ester, peptide, disulphide, silane, nucleoside, abasic nucleoside, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, polyhydrocarbon, phosphate ester, phosphoramidate, thiophosphate, alkylphosphate , or photolabile linker.
  • the linker is a biodegradable linker.
  • cationic salt refers to any organic or inorganic salt having a net positive charge, for example a triethylammonium (TEA) salt.
  • degradable linker refers to linker moieties that are capable of cleavage under various conditions. Conditions suitable for cleavage can include but are not limited to pH, UV irradiation, enzymatic activity, temperature, hydrolysis, elimination, and substitution reactions, and thermodynamic properties of the linkage.
  • photolabile linker refers to linker moieties as are known in the art, that are selectively cleaved under particular UV wavelengths.
  • lipid refers to any lipophilic compound.
  • Non- limiting examples of lipid compounds include fatty acids and their derivatives, including straight chain, branched chain, saturated and unsaturated fatty acids, carotenoids, terpenes, bile acids, and steroids, including cholesterol and derivatives or analogs thereof.
  • folic acid refers to analogs and derivatives of folic acid, for example antifolates, dihydrofloates, tetrahydrofolates, tetrahydorpterins, folinic acid, pteropolyglutamic acid, 1-deza, 3-deaza, 5-deaza, 8-deaza, 10-deaza, 1,5-deaza, 5,10 dideaza, 8,10-dideaza, and 5,8-dideaza folates, antifolates, and pteroic acid derivatives.
  • compounds with neutral charge refers to compositions which are neutral or uncharged at neutral or physiological pH.
  • Examples of such compounds are cholesterol and other steroids, cholesteryl hemisuccinate (CHEMS), dioleoyl phosphatidyl choline, distearoylphosphotidyl choline (DSPC), fatty acids such as oleic acid, phosphatidic acid and its derivatives, phosphatidyl serine, polyethylene glycol -conjugated phosphatidylamine, phosphatidylcholine, phosphatidylethanolamine and related variants, prenylated compounds including farnesol, polyprenols, tocopherol, and their modified forms, diacylsuccinyl glycerols, fusogenic or pore forming peptides, dioleoylphosphotidylethanolamine (DOPE), ceramide and the like.
  • CHEMS cholesteryl hemisuccinate
  • DSPC distearoylphosphotidyl choline
  • fatty acids such as oleic acid
  • lipid aggregate refers to a lipid-containing composition wherein the lipid is in the form of a liposome, micelle (non-lamellar phase) or other aggregates with one or more lipids.
  • biological system refers to a eukaryotic system or a prokaryotic system, can be a bacterial cell, plant cell or a mammalian cell, or can be of plant origin, mammalian origin, yeast origin, Drosophila origin, or archebacterial origin.
  • systemic administration refers to the in vivo systemic absorption or accumulation of drugs in the blood stream followed by distribution throughout the entire body.
  • Administration routes which lead to systemic absorption include, without limitations: intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular.
  • Each of these administration routes exposes the desired negatively charged polymers, e.g., nucleic acids, to an accessible diseased tissue.
  • the rate of entry of a drug into the circulation has been shown to be a function of molecular weight or size.
  • the use of a liposome or other drug carrier comprising the compounds of the instant invention can potentially localize the drug, for example, in certain tissue types, such as the tissues of the reticular endothelial system (RES).
  • RES reticular endothelial system
  • a liposome formulation which can facilitate the association of drug with the surface of cells, such as, lymphocytes and macrophages are also useful. This approach can provide enhanced delivery of the drug to target cells by taking advantage of the specificity of macrophage and lymphocyte immune recognition of abnormal cells, such as the cancer cells.
  • compositions or pharmaceutical formulation refers to a composition or formulation in a form suitable for administration, for example, systemic administration, into a cell or patient, preferably a human. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, or by injection. Such forms should not prevent the composition or formulation to reach a target cell (i.e, a cell to which the negatively charged polymer is targeted).
  • target cell i.e, a cell to which the negatively charged polymer is targeted.
  • Figure 1 shows non-limiting examples of cationic delivery reagents of the invention.
  • Diamine (1) can be converted to a bis-guanidinium derivative (2), or alternately a guanidinium derivative (3), that can be further conjugated with a ligand (L) to generate (4).
  • Any of compounds 1-4 can be complexed with a compound of interest, such as a nucleic acid or siNA construct, to generate a composition to facilitate the cellular delivery of the compound of interest.
  • a compound of interest such as a nucleic acid or siNA construct
  • Other diamines having differing alkyl chain lengths can be similarly used to generate a variety of diamino, bis-guanidinium, guanidinium, and ligand derivatized complexes.
  • FIG. 2 shows non-limiting examples of cationic delivery reagents of the invention.
  • Diamine (5) can be converted to a bis-guanidinium derivative (6), or alternately a guanidinium derivative (7), that can be further conjugated with a ligand (L) to generate (8).
  • Any of compounds 5-8 can be complexed with a compound of interest, such as a nucleic acid or siNA construct, to generate a composition to facilitate the cellular delivery of the compound of interest.
  • a compound of interest such as a nucleic acid or siNA construct
  • Other diamines having differing alkyl chain lengths or glycol composition can be similarly used to generate a variety of diamino, bis-guanidinium, guanidinium, and ligand derivatized complexes.
  • FIG. 3 shows non-limiting examples of cationic delivery reagents of the invention.
  • Diamine (9) can be converted to a bis-guanidinium derivative (10), or alternately a guanidinium derivative (11), that can be further conjugated with a ligand (L) to generate (12).
  • Any of compounds 9-12 can be complexed with a compound of interest, such as a nucleic acid or siNA construct, to generate a composition to facilitate the cellular delivery of the compound of interest.
  • a compound of interest such as a nucleic acid or siNA construct
  • Other diamine ethers having differing alkyl chain lengths can be similarly used to generate a variety of diamino, bis-guanidinium, guanidinium, and ligand derivatized complexes.
  • Figure 4 shows non-limiting examples of cationic delivery reagents of the invention.
  • Polyamine (13) can be converted to a bis-guanidinium derivative (14), or alternately a guanidinium derivative (15), that can be further conjugated with a ligand (L) to generate (16).
  • Any of compounds 13-16 can be complexed with a compound of interest, such as a nucleic acid or siNA construct, to generate a composition to facilitate the cellular delivery of the compound of interest.
  • a compound of interest such as a nucleic acid or siNA construct
  • Other polyamines having differing alkyl chain lengths and nitrogen content can be similarly used to generate a variety of diamino, bis-guanidinium, guanidinium, and ligand derivatized complexes.
  • Figure 5 shows non-limiting examples of cationic delivery reagents of the invention.
  • Spermidine (17) can be converted to a bis-guanidinium derivative (18), or alternately a guanidinium derivative (19), that can be further conjugated with a ligand (L) to generate (20).
  • Any of compounds 17-20 can be complexed with a compound of interest, such as a nucleic acid or siNA construct, to generate a composition to facilitate the cellular delivery of the compound of interest.
  • a compound of interest such as a nucleic acid or siNA construct
  • Other polyamines having differing alkyl chain lengths and nitrogen content, such as spermine can be similarly used to generate a variety of diamino, bis-guanidinium, guanidinium, and ligand derivatized complexes.
  • FIG. 6 shows non-limiting examples of cationic delivery reagents of the invention.
  • Tris-(2-aminoethyl)amine (TREN) (21) can be converted to a tri- guanidinium derivative (22), bis-guanidinium derivative (24), or alternately guanidinium derivative (23).
  • Compounds (23) and (24) can be further conjugated with a ligand (L) to generate compounds (25) and (26), and compound (25) can be further conjugated with the same or a different ligand to generate compound (27).
  • Any of compounds 21-27 can be complexed with a compound of interest, such as a nucleic acid or siNA construct, to generate a composition to facilitate the cellular delivery of the compound of interest.
  • a compound of interest such as a nucleic acid or siNA construct
  • FIG. 7 shows a non-limiting example of the synthesis of a spermidine based conjugate of the invention.
  • Spermine (17) is converted to a bis-guanidinium derivative (18) using di-Boc pyrazole carboxamidine.
  • Compound (18) is then coupled with a ligand (L), for via an amide linkage, to generate compound (28).
  • Compound (28) can be complexed with a compound of interest, such as a nucleic acid or siNA construct, to generate a composition to facilitate the cellular delivery of the compound of interest.
  • a compound of interest such as a nucleic acid or siNA construct
  • Other polyamines having differing alkyl chain lengths and nitrogen content can be similarly used to generate a variety of such polyamine ligand derivatized complexes.
  • Figure 8 shows a non-limiting example of the synthesis of an EDTA based conjugate of the invention.
  • a diamine such as diaminopropane (29) is coupled to a ligand, for example via an amide linkage, for generate compound (30) bearing a free amine.
  • Compound (30) is then coupled with EDTA to generate compound (31), which is then coupled with a polyamine, such as compound (18), to generate compound (32), bearing one, two, or three bis-guanidinium substituents. ).
  • Compound (32) can be complexed with a compound of interest, such as a nucleic acid or siNA construct, to generate a composition to facilitate the cellular delivery of the compound of interest.
  • Other polyamines having differing alkyl chain lengths and nitrogen content can be similarly used to generate a variety of such polyamine ligand derivatized complexes.
  • Figure 9 shows a non-limiting example of the synthesis of a 4-N-(Cholesterol- PEG)-Spermidine conjugate of the invention.
  • Figure 10 shows a non-limiting example of the synthesis of a 4-N- (Cholesterol-PEG)-Spermidyl-Bis-guanidine conjugate ofthe invention.
  • Figure 11 shows non-limiting examples of different stabilization chemistries
  • the compounds (e.g. compounds having any for Formulae 1-60 and/or biologically active molecules) of the instant invention can be used to administer pharmaceutical agents, such as biologically active molecules described herein.
  • Pharmaceutical agents prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all ofthe symptoms) of a disease state in a patient.
  • the compounds (e.g. compounds having any for Formulae 1-60 and/or biologically active molecules) of the instant invention are introduced by any standard means, with or without stabilizers, buffers, and the like, to form a composition.
  • a liposome delivery mechanism standard protocols for formation of liposomes can be followed.
  • the compositions of the present invention can also be formulated and used as tablets, capsules or elixirs for oral administration; suppositories for rectal administration; sterile solutions; suspensions for injectable administration; and the like.
  • the present invention also includes pharmaceutically acceptable formulations of the compounds described above, preferably in combination with the molecule(s) to be delivered.
  • formulations include salts of the above compounds, e.g., acid addition salts, for example, salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid.
  • the invention features the use of the compounds of the invention in a composition comprising surface-modified liposomes containing poly (ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes).
  • the invention features the use of compounds of the invention covalently attached to polyethylene glycol.
  • compositions have been shown to accumulate selectively in tumors, presumably by extravasation and capture in the neovascularized target tissues (Lasic et al. Science 1995, 267, 1275- 1276; Oku et al,1995, Biochim. Biophys. Acta, 1238, 86-90).
  • the long-circulating compositions enhance the pharmacokinetics and pharmacodynamics of therapeutic compounds, such as DNA and RNA, particularly compared to conventional cationic liposomes which are known to accumulate in tissues of the MPS (Liu et al, J. Biol. Chem.
  • compositions are also likely to protect drugs from nuclease degradation to a greater extent compared to cationic liposomes, based on their ability to avoid accumulation in metabolically aggressive MPS tissues such as the liver and spleen.
  • the present invention also includes a composition(s) prepared for storage or administration that includes a pharmaceutically effective amount of the desired compound(s) in a pharmaceutically acceptable carrier or diluent.
  • Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro edit. 1985) hereby incorporated by reference herein.
  • preservatives, stabilizers, dyes and flavoring agents can be included in the composition. Examples of such agents include but are not limited to sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid.
  • antioxidants and suspending agents can be included in the composition.
  • a pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state.
  • the pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concu ⁇ ent medication, and other factors which those skilled in the medical arts will recognize. Generally, an amount between 0.1 mg kg and 100 mg/kg body weight/day of active ingredients is administered dependent upon potency of the negatively charged polymer. Furthermore, the compounds of the invention and formulations thereof can be administered to a fetus via administration to the mother of a fetus.
  • the compounds of the invention and formulations thereof can be administered orally, topically, parenterally, by inhalation or spray or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles.
  • parenteral as used herein includes percutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, or intrathecal injection or infusion techniques and the like.
  • a pharmaceutical formulation comprising a nucleic acid molecule of the invention and a pharmaceutically acceptable carrier.
  • One or more nucleic acid molecules of the invention can be present in association with one or more non-toxic pharmaceutically acceptable carriers and/or diluents and/or adjuvants, and if desired other active ingredients.
  • compositions containing nucleic acid molecules of the invention can be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs.
  • compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more such sweetening agents, flavoring agents, coloring agents or preservative agents in order to provide pharmaceutically elegant and palatable preparations.
  • Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable exeipients that are suitable for the manufacture of tablets.
  • These exeipients can be, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc.
  • the tablets can be uncoated or they can be coated by known techniques. In some cases such coatings can be prepared by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as glyceryl monosterate or glyceryl distearate can be employed.
  • Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.
  • Aqueous suspensions contain the active materials in admixture with exeipients suitable for the manufacture of aqueous suspensions.
  • Such exeipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropyl- methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents can be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate.
  • dispersing or wetting agents can be a naturally-occurring phosphatide, for example, lec
  • the aqueous suspensions can also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.
  • Oily suspensions can be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.
  • the oily suspensions can contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents and flavoring agents can be added to provide palatable oral preparations.
  • compositions can be preserved by the addition of an anti-oxidant such as ascorbic acid.
  • Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents or suspending agents are exemplified by those already mentioned above. Additional exeipients, for example sweetening, flavoring and coloring agents, can also be present.
  • compositions of the invention can also be in the form of oil-in- water emulsions.
  • the oily phase can be a vegetable oil or a mineral oil or mixtures of these.
  • Suitable emulsifying agents can be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example, sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate.
  • the emulsions can also contain sweetening and flavoring agents.
  • Syrups and elixirs can be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol, glucose or sucrose. Such formulations can also contain a demulcent, a preservative and flavoring and coloring agents.
  • the pharmaceutical compositions can be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above.
  • the sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol.
  • Suitable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono-or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • the compounds of the invention can also be administered in the form of suppositories, e.g., for rectal administration of the drug.
  • suppositories e.g., for rectal administration of the drug.
  • These compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • Such materials include cocoa butter and polyethylene glycols.
  • Compounds of the invention can be administered parenterally in a sterile medium.
  • the drug depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle.
  • adjuvants such as local anesthetics, preservatives and buffering agents can be dissolved in the vehicle.
  • Dosage levels of the order of from about 0.1 mg to about 140 mg per kilogram of body weight per day are useful in the treatment of the above-indicated conditions (about 0.5 mg to about 7 g per patient per day).
  • the amount of active ingredient that can be combined with the ca ⁇ ier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. Dosage unit forms will generally contain between from about 1 mg to about 500 mg of an active ingredient. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy.
  • the composition can also be added to the animal feed or drinking water. It can be convenient to formulate the animal feed and drinking water compositions so that the animal takes in a therapeutically appropriate quantity of the composition along with its diet. It can also be convenient to present the composition as a premix for addition to the feed or drinking water.
  • the compounds of the present invention can also be administered to a patient in combination with other therapeutic compounds to increase the overall therapeutic effect. The use of multiple compounds to treat an indication can increase the beneficial effects while reducing the presence of side effects.
  • small nucleic acid motifs refers to nucleic acid motifs no more than 100 nucleotides in length, preferably no more than 80 nucleotides in length, and most preferably no more than 50 nucleotides in length; (e.g., individual siNA oligonucleotide sequences or siNA sequences synthesized in tandem) are preferably used for exogenous delivery.
  • the simple structure of these molecules increases the ability of the nucleic acid to invade targeted regions of protein and/or RNA structure.
  • Exemplary molecules of the instant invention are chemically synthesized, and others can similarly be synthesized.
  • Oligonucleotides are synthesized using protocols known in the art, for example as described in Caruthers et al, 1992, Methods in Enzymology 21 1, 3-19, Thompson et al, International PCT Publication No. WO 99/54459, Wincott et al, 1995, Nucleic Acids Res. 23, 2677-2684, Wincott et al, 1997, Methods Mol. Bio., 74, 59, Brennan et al, 1998, Biotechnol Bioeng, 61, 33-45, and Brennan, U.S. Pat. No. 6,001,311.
  • oligonucleotides makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end.
  • small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 ⁇ mol scale protocol with a 2.5 minute coupling step for 2'-0-methylated nucleotides and a 45 second coupling step for 2'-deoxy nucleotides or 2'-deoxy-2'-fluoro nucleotides.
  • Table I outlines the amounts and the contact times of the reagents used in the synthesis cycle.
  • syntheses at the 0.2 ⁇ mol scale can be performed on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, CA) with minimal modification to the cycle.
  • synthesizer include the following: detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); and oxidation solution is 16.9 mM 12, 49 mM pyridine, 9% water in THF (PERSEPTIVETM). Burdick & Jackson Synthesis Grade acetonitrile is used directly from the reagent bottle. S -Ethyl tetrazole solution (0.25 M in acetonitrile) is made up from the solid obtained from American International Chemical, Inc. Alternately, for the introduction of phosphorothioate linkages, Beaucage reagent (3H-l ,2-Benzodithiol-3-one 1,1- dioxide, 0.05 M in acetonitrile) is used.
  • Deprotection of the D ⁇ A-based oligonucleotides is performed as follows: the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 40% aqueous methylamine (1 mL) at 65 °C for 10 minutes. After cooling to -20 °C, the supernatant is removed from the polymer support. The support is washed three times with 1.0 mL of EtOH:MeC ⁇ :H20/3 : 1 : 1 , vortexed and the supernatant is then added to the first supernatant.
  • RNA including certain siNA molecules of the invention follows the procedure as described in Usman et al, 1987, J. Am. Chem. Soc, 109, 7845; Scaringe et al, 1990, Nucleic Acids Res., 18, 5433; and Wincott et al, 1995, Nucleic Acids Res. 23, 2677-2684 Wincott et al, 1997, Methods Mol Bio., 74, 59, and makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end.
  • small scale syntheses are conducted on a 394 Applied Biosystems, Inc. synthesizer using a 0.2 ⁇ mol scale protocol with a 7.5 minute coupling step for alkylsilyl protected nucleotides and a 2.5 minute coupling step for 2'-0-methylated nucleotides.
  • Table I outlines the amounts and the contact times of the reagents used in the synthesis cycle.
  • syntheses at the 0.2 ⁇ mol scale can be done on a 96-well plate synthesizer, such as the instrument produced by Protogene (Palo Alto, CA) with minimal modification to the cycle.
  • Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer, determined by colorimetric quantitation of the trityl fractions, are typically 97.5-99%.
  • synthesizer include the following: detritylation solution is 3% TCA in methylene chloride (ABI); capping is performed with 16% N- methyl imidazole in THF (ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); oxidation solution is 16.9 mM 12, 49 mM pyridine, 9% water in THF
  • Deprotection and purification of the si ⁇ A can be performed as is generally described in Usman et al, US 5,831,071, US 6,353,098, US 6,437,117, and
  • oligonucleotides comprising 2'-deoxy-2'-fluoro nucleotides can degrade under inappropriate deprotection conditions. Such oligonucleotides are deprotected using aqueous methylamine at about 35°C for 30 minutes.
  • the 2'-deoxy-2'-fluoro containing oligonucleotide also comprises ribonucleotides
  • TEA-HF is added and the reaction maintained at about 65°C for an additional 15 minutes.
  • the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass screw top vial and suspended in a solution of 33% ethanolic methylamine/DMSO: 1/1 (0.8 mL) at 65 °C for 15 minutes.
  • the vial is brought to room temperature.
  • TEA «3HF (0.1 mL) is added and the vial is heated at 65 °C for 15 minutes.
  • the sample is cooled at -20 °C and then quenched with 1.5 M NH4HCO3.
  • the quenched NH4HCO3 solution is loaded onto a C-18 containing cartridge that had been prewashed with acetonitrile followed by 50 mM TEAA. After washing the loaded cartridge with water, the RNA is detritylated with 0.5% TFA for 13 minutes. The cartridge is then washed again with water, salt exchanged with 1 M NaCI and washed with water again. The oligonucleotide is then eluted with 30% acetonitrile.
  • the average stepwise coupling yields are typically >98% (Wincott et al, 1995 Nucleic Acids Res. 23, 2677-2684).
  • the scale of synthesis can be adapted to be larger or smaller than the example described above including but not limited to 96-well format.
  • nucleic acid molecules of the present invention can be synthesized separately and joined together post-synthetically, for example, by ligation (Moore et al, 1992, Science 256, 9923; Draper et al, International PCT publication No. WO 93/23569; Shabarova et al, 1991, Nucleic Acids Research 19, 4247; Bellon et al, 1997, Nucleosides & Nucleotides, 16, 951; Bellon et al, 1997, Bioconjugate Chem. 8, 204), or by hybridization following synthesis and/or deprotection.
  • the nucleic acid molecules (e.g. siNA molecules) of the invention can also be synthesized via a tandem synthesis methodology, wherein both siNA strands are synthesized as a single contiguous oligonucleotide fragment or strand separated by a cleavable linker which is subsequently cleaved to provide separate siNA fragments or strands that hybridize and permit purification of the siNA duplex (see McSwiggen et al, USSN (10/444,853), filed May 23, 2003).
  • the linker can be a polynucleotide linker or a non-nucleotide linker.
  • tandem synthesis of siNA as described herein can be readily adapted to both multiwell/multiplate synthesis platforms such as 96 well or similarly larger multi-well platforms.
  • the tandem synthesis of siNA as described herein can also be readily adapted to large scale synthesis platforms employing batch reactors, synthesis columns and the like.
  • a nucleic acid molecule e.g. siNA molecule
  • nucleic acid molecules of the present invention can be modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-fluoro, 2'-O-methyl, 2'-H (for a review see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al, 1994, Nucleic Acids Symp. Ser. 31, 163).
  • siNA constructs can be purified by gel electrophoresis using general methods or can be purified by high pressure liquid chromatography (HPLC; see Wincott et al, supra, the totality of which is hereby incorporated herein by reference) and re- suspended in water. Optimizing Activity ofthe nucleic acid molecules of the invention.
  • oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-O- methyl, 2'-H, nucleotide base modifications (for a review see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al, 1994, Nucleic Acids Symp. Ser. 31, 163; Burgin et al, 1996, Biochemistry , 35, 14090).
  • nuclease resistant groups for example, 2'-amino, 2'-C-allyl, 2'-flouro, 2'-O- methyl, 2'-H, nucleotide base modifications
  • nucleic acid molecules having chemical modifications that maintain or enhance activity are provided. Such nucleic acid is also generally more resistant to nucleases than unmodified nucleic acid. Thus, in a cell and/or in vivo the activity can not be significantly lowered.
  • Therapeutic nucleic acid molecules e.g., enzymatic nucleic acid molecules and antisense nucleic acid molecules delivered exogenously are optimally stable within cells until translation of the target RNA has been inhibited long enough to reduce the levels of the undesirable protein.
  • RNA and DNA have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability as described above.
  • nucleic acid-based molecules of the invention can lead to better treatment of the disease progression by affording the possibility of combination therapies (e.g., multiple antisense or enzymatic nucleic acid molecules targeted to different genes, nucleic acid molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations of molecules (including different motifs) and/or other chemical or biological molecules).
  • combination therapies e.g., multiple antisense or enzymatic nucleic acid molecules targeted to different genes, nucleic acid molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations of molecules (including different motifs) and/or other chemical or biological molecules).
  • the treatment of patients with nucleic acid molecules can also include combinations of different types of nucleic acid molecules.
  • nucleic acid molecules having chemical modifications that maintain or enhance biologic activity are provided.
  • Such nucleic acids are also generally more resistant to nucleases than unmodified nucleic acid.
  • the activity of the nucleic acid can not be significantly lowered.
  • enzymatic nucleic acids are useful in a cell and/or in vivo even if activity over all is reduced 10 fold (Burgin et al, 1996, Biochemistry, 35, 14090).
  • Such nucleic acids herein are said to "maintain” the activity of an all RNA or all DNA nucleic acid molecule (e.g. siNA, antisense, or enzymatic nucleic acid molecule).
  • the nucleic acid molecules comprise a 5' and/or a 3'- cap structure.
  • the 3 '-cap includes, for example 4',5'-methylene nucleotide; 1 -(beta-D-erythrofuranosyl) nucleotide; 4'-thio nucleotide, carbocyclic nucleotide; 5'-amino-alkyl phosphate; l,3-diamino-2-propyl phosphate, 3- aminopropyl phosphate; 6-aminohexyl phosphate; 1 ,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide; alpha- nucleotide; modified base nucleotide; phosphorodithioate; tbreo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-
  • the invention features modified nucleic acid molecules with phosphate backbone modifications comprising one or more phosphorothioate, phosphorodithioate, methylphosphonate, morpholino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl, substitutions.
  • amino is meant 2'-NH 2 or 2'-O- NH 2 , which can be modified or unmodified. Such modified groups are described, for example, in Eckstein et al, U.S. Patent 5,672,695 and Matulic-Adamic et al, W ⁇ 98/28317, respectively, which are both incorporated by reference in their entireties. Various modifications to nucleic acid (e.g., siNA, antisense and ribozyme) structure can be made to enhance the utility of these molecules.
  • nucleic acid e.g., siNA, antisense and ribozyme
  • such modifications can enhance shelf-life, half-life in vitro, stability, and ease of introduction of such oligonucleotides to the target site, including e.g., enhancing penetration of cellular membranes and conferring the ability to recognize and bind to targeted cells.
  • nucleic acid molecules can lead to better treatment of disease progression by affording the possibility of combination therapies (e.g., multiple nucleic acid molecules targeted to different genes, nucleic acid molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations of nucleic acid molecules (e.g. siNA, antisense, ribozymes, aptamers etc.) and/or other chemical or biological molecules).
  • the treatment of patients with nucleic acid molecules can also include combinations of different types of nucleic acid molecules.
  • Therapies can be devised which include a mixture of nucleic acid molecules (e.g. siNA, antisense, ribozymes, aptamers etc), to one or more targets to alleviate symptoms of a disease.
  • cancers and cancerous conditions such as breast, lung, prostate, colorectal, brain, esophageal, stomach, bladder, pancreatic, cervical, hepatocellular, head and neck, and ovarian cancer, melanoma, lymphoma, glioma, multidrug resistant cancers; ocular conditions such as macular degeneration and diabetic retinopathy, and/or viral infections including HIV, HBV, HCV, CMV, RSV, HSV, poliovirus, influenza, rhinovirus, west nile virus, severe acute respiratory syndrome (SARS) virus, Ebola virus, foot and mouth virus, papilloma virus, and/or SARS virus infection.
  • cancers and cancerous conditions such as breast, lung, prostate, colorectal, brain, esophageal, stomach, bladder, pancreatic, cervical, hepatocellular, head and neck, and ovarian cancer, melanoma, lymphoma, glioma, multidrug resistant cancers
  • the molecules of the invention can be used in conjunction with other known methods, therapies, or drugs.
  • monoclonal antibodies eg; mAb IMC C225, mAB ABX-EGF
  • TKIs tyrosine kinase inhibitors
  • OSI-774 and ZD1839 tyrosine kinase inhibitors
  • chemotherapy and/or radiation therapy
  • chemotherapies that can be combined with nucleic acid molecules of the instant invention include various combinations of cytotoxic drugs to kill the cancer cells.
  • These drugs include, but are not limited to, paclitaxel (Taxol), docetaxel, cisplatin, methotrexate, cyclophosphamide, doxorubin, fluorouracil carboplatin, edatrexate, gemcitabine, vinorelbine etc.
  • paclitaxel Taxol
  • docetaxel cisplatin
  • methotrexate cyclophosphamide
  • doxorubin fluorouracil carboplatin
  • edatrexate gemcitabine
  • vinorelbine vinorelbine
  • N, N'- bis(tert-butoxycarbonyl) protected guanidinium intermediates are then suspended in anhydrous methanol a solution of 4.0 M hydrogen chloride in 1,4- dioxane is added and the resulting gas is liberated from the reaction. The resulting solution is stirred at 40 °C overnight and is then concentrated under vacuum to yield a solid, which is reconstituted in anhydrous methanol. Guanidinium compounds (3), (7), (11), (15), (19), or (23) are then obtained by crystallization, for example in dichloromethane/methanol.
  • Tris(2-aminoethyl)amine (21) (1.50 mL, 10 mmol) was co-evaporated with 1,4- dioxane (2 x 10 mL), then dissolved in anhydrous 1 ,4-dioxane.
  • N-tert- butoxycarbonyl-/H-pyrazole-l-carboxamidine (7.56 g, 36 mmol) and TEA (5.0 mL, 36 mmol) were added.
  • the Cholesterol-PEG-NHS ester (33) (0J9 g, 1.08 mmol) was dissolved in anhydrous dichloromethane (10 mL).
  • the l,8-di-(N,N'-bis-Boc-guanidinium)- permidine (37) (0.885 g, 1.3 eq.) and DIPEA (0.47 mL, 2.5 eq.) were added.
  • the reaction mixture was stined at room temperature overnight, and then poured into sodium bicarbonate (5% aqueous, 80 mL).
  • the product was extracted with dichloromethane (80 mL).
  • the organic layer was washed with sodium bicarbonate (5%) once, dried with sodium sulfate, and concentrated.
  • the resulting residue was chromatographed on silica gel (50% ethyl acetate/ DCM) to give 0.96 g (72%) of product (39) as a white foam.
  • ES-MS 1246.2
  • a si ⁇ A molecule such as a si ⁇ A duplex, is complexed with a cationic compound based upon charge ratio.
  • the complex can be formulated with different charge ratios by using equivalents of nucleic acid to cation to generate a formulation with a net positive charge (e.g. excess cation to nucleic acid), a neutral charge, or a net negative charge (e.g. excess nucleic acid to cation).
  • the cation can be titrated into a solution of nucleic acid or the nucleic acid can be titrated into a solution of the cationic compound.
  • AGGUGAAGCGAAGMGCACATST wherein A and G are 2'-0-methyl nucleotides and u and c are 2'-deoxy-2'-fluoro nucleotides (SEQ ID No: 2)) was obtained in HPLC purified form and dissolved in sterile Milli-Q water to a concentration of 895uM (approximately 15 mg siNA/ml water). Because there are 42 phosphates per mole of duplex siNA, the net polyanion charge of the duplex is calculated at 37.6mM in this solution. A lOOuL aliquot of the siNA solution contains 3.8 micromoles of phosphate anion.
  • the resulting solution was filtered to 0.2 micron absolute using cell culture grade disposable filters prior to use.
  • the solutions were stored at 5-8 C prior to use. All solutions remained free of precipitates during storage.
  • Additional instrumental techniques performed to characterize the cationic complexes included static light scattering and size exclusion chromatography using a Wyatt Technologies miniDawn detector with additional QELS hardware and Astra software (Wyatt Tecnologies, Santa Barbera, CA).
  • the size exclusion chromatography was performed using a TosoHaas TSK-gel SWxl column (4mm x 300mm) and Agilent 1100 HPLC hardware including binary pump G1312A and RJD detector G1362A plus Chemstation software A.08.03.
  • This instrument can detect and quantitate hydrodynamic size of the cationic nucleic acid complexes and provide information on extinction coefficients of the nucleic acid component and molecular weight information of the complex through use of Zimm Rouse equations for light scattering.
  • a Brookhaven Instrument Corporation ZetaPALS dynamic light scattering instrument was used to measure size distributions of the cationic nucleic acid complexes and characterize the zeta-potential of these complexes. The data collected for all complexes made to date shows a predominant fraction of small monomeric particles for these complexes. A large shift in zeta potential towards positive numbers was observed for all nucleic acid complexes containing cationic amines.
  • the starting siRNA duplex material had a negative zeta potential as is always observed for the polyanionic nucleic acids.
  • a positive zeta-potential is a strong indication that the cationic amine has complexed the nucleic acids and created a new particle with a different net charge than the starting material.
  • the cationic compounds of the invention can be formulated into a lipoplex comprising a cationic component, a lipid component, and a biologically active molecule component (e.g. siNA).
  • a lipoplex can lead to improved pharmacokinetic properties such as increased half life and increased serum stability of biologically active molecules to be delivered to relevant cells and tissues.
  • a standard neutral phosphatidylethanolamine lipid was purchased from Avanti Polar Lipids as a 10 mg/mL solution in chloroform (Avanti Cat. No. 850402, 1,2-Diphytanoyl-sn-Glycero- 3-Phosphoethanolamine, F.W.
  • a cationic amine conjugated to cholesterol via a tetraethylene glycol ether linkage (compound 36, Figure 9) was prepared as described herein. 550uL of the Cholesterol conjugate at 20 mg/mL in chloroform was added to 900 uL of DPhPE neutral lipid at 10 mg/mL and the solution was evaporated to dryness on a Buichi rotary evaporator with a water bath temperature of 25C. The flask containing the film of lipids in a 1 : 1 stoichiometric ratio was placed on a vacuum manifold and pumped overnight with a belt driven rotary vane vacuum pump to remove residual chloroform solvent.
  • the dry lipid film was re-hydrated for 2 hours with the addition of 2 mL of sterile water and brief periods of sonication (2 x 10 minutes each period to prevent overheating of the lipoplex formulation).
  • the sonication was followed by particle size measurement with a Brookhaven Instruments Zeta-PALLS instrument.
  • the light scattering data showed the presence of a monodisperse particle with an effective diameter of 107 nm.
  • siNA to lipoplex charge ratio was titrated between 1 :2, 1 :3, 1 :4, and 1 :5 mole equivalents of siNA phosphate to compound (36). These titration experiments resulted in a lipoplex with overall net positive charge in all cases (20% of cationic sites occupied at 1 :5 ratio with siNA phosphates to 50% occupation at 1 :2 ratio) and these results were confirmed with zeta potential measurement using the BIC zeta- PALLS instrument. All formulations yielded a positive Zeta potential after complexing siNA at the previously described ratios of siNA to lipoplex. To generate a 1 to 2 ratio of siNA to lipoplex required the following concentrations and volume of lipoplex and siNA solutions.
  • siNA siNA molecules can be designed to interact with various sites in the RNA message, for example, target sequences within the RNA sequences described herein.
  • the sequence of one strand of the siNA molecule(s) is complementary to the target site sequences described above.
  • the siNA molecules can be chemically synthesized using methods described herein.
  • Inactive siNA molecules that are used as control sequences can be synthesized by scrambling the sequence of the siNA molecules such that it is not complementary to the target sequence.
  • siNA constructs can by synthesized using solid phase oligonucleotide synthesis methods as described herein (see for example Usman et al, US Patent Nos.
  • RNA oligonucleotides are synthesized in a stepwise fashion using the phosphoramidite chemistry as is known in the art.
  • Standard phosphoramidite chemistry involves the use of nucleosides comprising any of 5'-0- dimethoxytrityl, 2'-0-tert-butyldimethylsilyl, 3'-0-2-Cyanoethyl N,N- diisopropylphos-phoroamidite groups, and exocyclic amine protecting groups (e.g. N6-benzoyl adenosine, N4 acetyl cytidine, and N2-isobutyryl guanosine).
  • 2'-0-Silyl Ethers can be used in conjunction with acid-labile 2'-0-orthoester protecting groups in the synthesis of RNA as described by Scaringe supra.
  • Differing 2' chemistries can require different protecting groups, for example 2'-deoxy-2'-amino nucleosides can utilize N-phthaloyl protection as described by Usman et al, US Patent 5,631,360, incorporated by reference herein in its entirety).
  • each nucleotide is added sequentially (3 '- to 5'- direction) to the solid support-bound oligonucleotide.
  • the first nucleoside at the 3'- end of the chain is covalently attached to a solid support (e.g., controlled pore glass or polystyrene) using various linkers.
  • the nucleotide precursor, a ribonucleoside phosphoramidite, and activator are combined resulting in the coupling of the second nucleoside phosphoramidite onto the 5 '-end of the first nucleoside.
  • the support is then washed and any unreacted 5 '-hydroxyl groups are capped with a capping reagent such as acetic anhydride to yield inactive 5 '-acetyl moieties.
  • a capping reagent such as acetic anhydride to yield inactive 5 '-acetyl moieties.
  • the trivalent phosphorus linkage is then oxidized to a more stable phosphate linkage.
  • the 5'-0-protecting group is cleaved under suitable conditions (e.g., acidic conditions for trityl-based groups and Fluoride for silyl-based groups). The cycle is repeated for each subsequent nucleotide.
  • Modification of synthesis conditions can be used to optimize coupling efficiency, for example by using differing coupling times, differing reagent/phosphoramidite concentrations, differing contact times, differing solid supports and solid support linker chemistries depending on the particular chemical composition of the siNA to be synthesized.
  • Deprotection and purification of the siNA can be performed as is generally described in Usman et al, US 5,831 ,071, US 6,353,098, US 6,437,1 17, and Bellon et al, US 6,054,576, US 6,162,909, US 6,303,773, or Scaringe supra, incorporated by reference herein in their entireties. Additionally, deprotection conditions can be modified to provide the best possible yield and purity of siNA constructs.
  • oligonucleotides comprising 2'-deoxy-2'-fluoro nucleotides can degrade under inappropriate deprotection conditions. Such oligonucleotides are deprotected using aqueous methylamine at about 35°C for 30 minutes. If the 2'-deoxy-2'-fluoro containing oligonucleotide also comprises ribonucleotides, after deprotection with aqueous methylamine at about 35°C for 30 minutes, TEA-HF is added and the reaction maintained at about 65°C for an additional 15 minutes.
  • Example 5 Nucleic acid inhibition of target RNA in vivo siNA molecules targeted to the target RNA are designed, synthesized, and formulated with polycationic delivery compounds as described above. These complexed nucleic acid molecules can be tested for cleavage activity in vivo, for example, using the following procedure. Two formats are used to test the efficacy of siNAs targeting a particular gene transcipt. First, the reagents are tested on target expressing cells (e.g., HeLa), to determine the extent of RNA and protein inhibition. siNA reagents are selected against the RNA target. RNA inhibition is measured after delivery of these reagents to cells using formulations of the invention.
  • target expressing cells e.g., HeLa
  • RNA inhibition Relative amounts of target RNA are measured versus actin using real-time PCR monitoring of amplification (eg., ABI 7700 Taqman®).
  • a comparison is made to a mixture of oligonucleotide sequences made to unrelated targets or to a randomized siNA control with the same overall length and chemistry, but with randomly substituted nucleotides at each position.
  • Primary and secondary lead reagents are chosen for the target and optimization performed. After an optimal transfection agent and concentration is chosen, a RNA time-course of inhibition is performed with the lead siNA molecule.
  • a cell-plating format can be used to determine RNA inhibition.
  • siNA Delivery of siNA to Cells
  • Cells e.g., HeLa
  • EGM-2 BioWhittaker
  • siNA final concentration, for example 20nM
  • cationic delivery agent e.g., final concentration 2Dg/ml
  • EGM basal media Biowhittaker
  • the complexed siNA is added to each well and incubated for the times indicated.
  • cells are seeded, for example, at 1x10 ⁇ in 96 well plates and siNA complex added as described.
  • Efficiency of delivery of siNA to cells is determined using a fluorescent siNA complexed with lipid.
  • Cells in 6-well dishes are incubated with siNA for 24 hours, rinsed with PBS and fixed in 2% paraformaldehyde for 15 minutes at room temperature. Uptake of siNA is visualized using a fluorescent microscope.
  • Total RNA is prepared from cells following siNA delivery, for example, using
  • RT-PCR amplifications are performed on, for example, an ABI PRISM 7700 Sequence Detector using 50 ⁇ l reactions consisting of 10 ⁇ l total RNA, 100 nM forward primer, 900 nM reverse primer, 100 nM probe, IX TaqMan PCR reaction buffer (PE-Applied Biosystems), 5.5 mM MgCl 2 , 300 ⁇ M each dATP, dCTP, dGTP, and dTTP, 10U RNase Inhibitor (Promega), 1.25U AmpliTaq Gold (PE- Applied Biosystems) and 10U M-MLV Reverse Transcriptase (Promega).
  • the thermal cycling conditions can consist of 30 min at 48°C, 10 min at 95°C, followed by 40 cycles of 15 sec at 95°C and 1 min at 60°C.
  • Quantitation of mRNA levels is determined relative to standards generated from serially diluted total cellular RNA (300, 100, 33, 1 1 ng/rxn) and normalizing to ⁇ -actin or GAPDH mRNA in parallel TaqMan reactions.
  • an upper and lower primer and a fluorescently labeled probe are designed.
  • Real time incorporation of SYBR Green I dye into a specific PCR product can be measured in glass capillary tubes using a lightcyler.
  • a standard curve is generated for each primer pair using control cRNA. Values are represented as relative expression to GAPDH in each sample.
  • Nuclear extracts can be prepared using a standard micro preparation technique (see for example Andrews and Faller, 1991 , Nucleic Acids Research, 19, 2499). Protein extracts from supernatants are prepared, for example using TCA precipitation. An equal volume of 20% TCA is added to the cell supernatant, incubated on ice for 1 hour and pelleted by centrifugation for 5 minutes. Pellets are washed in acetone, dried and resuspended in water. Cellular protein extracts are run on a 10% Bis-Tris NuPage (nuclear extracts) or 4-12% Tris-Glycine (supernatant extracts) polyacrylamide gel and transfened onto nitro-cellulose membranes.
  • Non-specific binding can be blocked by incubation, for example, with 5% non-fat milk for 1 hour followed by primary antibody for 16 hour at 4°C. Following washes, the secondary antibody is applied, for example (1: 10,000 dilution) for 1 hour at room temperature and the signal detected with SuperSignal reagent (Pierce).
  • Example 6 Animal Models Various animal models can be used to screen formulated siNA constructs in vivo as are known in the art, for example those animal models that are used to evaluate other nucleic acid technologies such as enzymatic nucleic acid molecules (ribozymes) and/or antisense. Such animal models are used to test the efficacy of formulated siNA molecules described herein. In a non-limiting example, siNA molecules that are designed as anti-angiogenic agents can be screened using animal models.
  • ribozymes enzymatic nucleic acid molecules
  • VEGFR e.g., VEGFR1, VEGFR2, and VEGFR3 genes.
  • VEGFR e.g., VEGFR1, VEGFR2, and VEGFR3 genes.
  • a comeal model has been used to study angiogenesis in rat and rabbit, since recruitment of vessels can easily be followed in this normally avascular tissue (Pandey et al, 1995 Science 268: 567-569).
  • angiogenesis factor e.g. bFGF or VEGF
  • Angiogenesis is monitored 3 to 5 days later.
  • siNA molecules directed against VEGFR mRNAs would be delivered in the disk as well, or dropwise to the eye over the time course of the experiment.
  • hypoxia has been shown to cause both increased expression of VEGF and neovascularization in the retina (Pierce et al, 1995 Proc. Natl. Acad. Sci. USA. 92: 905-909; Shweiki et ⁇ /., 1992 J. Clin. Invest. 91 : 2235-2243).
  • Several animal models exist for screening of anti-angiogenic agents include comeal vessel formation following comeal injury (Burger et al, 1985 Cornea 4: 35-41 ; Lepri, et al, 1994 J. Ocular Pharmacol.
  • This model involves an avascular tissue into which vessels are recruited by a stimulating agent (growth factor, thermal or alkalai burn, endotoxin).
  • a stimulating agent growth factor, thermal or alkalai burn, endotoxin.
  • the comeal model utilizes the intrastromal comeal implantation of a Teflon pellet soaked in a VEGF-Hydron solution to recruit blood vessels toward the pellet, which can be quantitated using standard microscopic and image analysis techniques.
  • siNA molecules are applied topically to the eye or bound within Hydron on the Teflon pellet itself.
  • This avascular cornea as well as the Matrigel model provide for low background assays. While the comeal model has been performed extensively in the rabbit, studies in the rat have also been conducted.
  • the mouse model (Passaniti et al, supra) is a non-tissue model which utilizes
  • Matrigel an extract of basement membrane (Kleinman et al, 1986) or Millipore® filter disk, which can be impregnated with growth factors and anti-angiogenic agents in a liquid form prior to injection.
  • the Matrigel or Millipore® filter disk forms a solid implant.
  • VEGF embedded in the Matrigel or Millipore® filter disk is used to recruit vessels within the matrix of the Matrigel or Millipore® filter disk which can be processed histologically for endothelial cell specific vWF (factor VITI antigen) immunohistochemistry,
  • the Matrigel Like the cornea, the Matrigel or
  • Millipore® filter disk are avascular; however, it is not tissue.
  • Millipore® filter disk model siNA molecules are administered within the matrix of the Matrigel or Millipore® filter disk to test their anti-angiogenic efficacy.
  • delivery issues in this model as with delivery of siNA molecules by Hydron- coated Teflon pellets in the rat cornea model, may be less problematic due to the homogeneous presence of the siNA within the respective matrix.
  • the Lewis lung carcinoma and B-16 murine melanoma models are well accepted models of primary and metastatic cancer and are used for initial screening of anti-cancer agents. These murine models are not dependent upon the use of immunodeficient mice, are relatively inexpensive, and minimize housing concerns. Both the Lewis lung and B-16 melanoma models involve subcutaneous implantation of approximately 10 ⁇ tumor cells from metastatically aggressive tumor cell lines (Lewis lung lines 3LL or D122, LLc-LN7; B-16-BL6 melanoma) in C57BL/6J mice. Alternatively, the Lewis lung model can be produced by the surgical implantation of tumor spheres (approximately 0.8 mm in diameter). Metastasis also may be modeled by injecting the tumor cells directly intraveneously.
  • systemic pharmacotherapy with a wide variety of agents usually begins 1-7 days following tumor implantation/inoculation with either continuous or multiple administration regimens.
  • Concunent pharmacokinetic studies can be performed to determine whether sufficient tissue levels of siNA can be achieved for pharmacodynamic effect to be expected.
  • primary tumors and secondary lung metastases can be removed and subjected to a variety of in vitro studies (i.e. target RNA reduction).
  • Ohno-Matsui et al, 2002, Am. J. Pathology, 160, 711-719 describe a model of severe proliferative retinopathy and retinal detachment in mice under inducible expression of vascular endothelial growth factor.
  • VEGFR1, VEGFR2, and/or VEGFR3 protein levels can be measured clinically or experimentally by FACS analysis.
  • VEGFR1, VEGFR2, and/or VEGFR3 encoded mRNA levels can be assessed by Northern analysis, RNase-protection, primer extension analysis and/or quantitative RT-PCR.
  • siNA molecules that block VEGFR1, VEGFR2, and/or VEGFR3 protein encoding mRNAs and therefore result in decreased levels of VEGFR1, VEGFR2, and/or VEGFR3 activity by more than 20% in vitro can be identified using the techniques described herein.
  • Example 7 Screening formulated siNA constructs for improved pharmacokinetics
  • formulated siNA constructs are screened in vivo for improved pharmacokinetic properties compared to siNA constructs that are not complexed with a delivery agent.
  • Lead siNA molecules are complexed with a delivery agent and the formulated constructs are tested in an appropriate system (e.g human serum for nuclease resistance, shown, or an animal model for PK/delivery parameters).
  • the formulated siNA construct is tested for RNAi activity, for example in a cell culture system such as a luciferase reporter assay).
  • Lead siNA formulations are then identified which possess a particular characteristic while maintaining RNAi activity, and can be further modified or optimized and assayed once again. This same approach can be used to identify siNA-conjugate molecules with improved pharmacokinetic profiles, delivery, localized delivery, cellular uptake, and RNAi activity.
  • Example 8 Inhibition of HCV RNA expression using complexed siNA formulations
  • Formulated siNA complexes with cationic polymers are tested for efficacy in reducing HCV RNA expression in, for example, Huh7 cells (see, for example, Randall et al, 2003, PNAS USA, 100, 235-240). Cells are plated approximately 24h before transfection in 96-well plates at 5,000-7,500 cells/well, 100 ⁇ l/well, such that at the time of transfection cells are 70-90% confluent.
  • annealed siNAs are complexed with a cationic polymer (see Example 2 above) in a volume of 50 ⁇ l/well and incubated for 20 minutes at room temperature.
  • siNA transfection mixtures are added to cells to give a final siNA concentration of 25 nM in a volume of 150 ⁇ l.
  • Each siNA transfection mixture is added to 3 wells for triplicate siNA treatments. Cells are incubated at 37° for 24h in the continued presence of the siNA transfection mixture.
  • RNA is prepared from each well of treated cells. The supematants with the transfection mixtures are first removed and discarded, then the cells are lysed and RNA prepared from each well.
  • Target gene expression following treatment is evaluated by RT-PCR for the target gene and for a control gene (36B4, an RNA polymerase subunit) for normalization. The triplicate data is averaged and the standard deviations determined for each treatment. Normalized data are graphed and the percent reduction of target mRNA by active siNAs in comparison to their respective inverted control siNAs is determined.
  • a nucleic acid molecule such as an enzymatic nucleic acid, antisense, or aptamer, is complexed with a cationic compound based upon charge ratio.
  • the complex can be formulated with different charge ratios by using equivalents of nucleic acid to cation to generate a formulation with a net positive charge (e.g. excess cation to nucleic acid), a neutral charge, or a net negative charge (e.g. excess nucleic acid to cation).
  • the cation can be titrated into a solution of nucleic acid or the nucleic acid can be titrated into a solution of the cationic compound.
  • the nucleic acid molecule is obtained in HPLC purified form and dissolved in sterile Milli-Q water.
  • a stock solution of cationic amine is added in a suitable amount based upon the number of phosphates present in the nucleic acid molecule. For example, to generate a formulation with a net positive charge, an excess molar equivalence is used.
  • the resulting solution is analyzed by strong anion exchange chromatography for concentration and purity. The solutions are then used in a cell culture assays to screen for efficacy of the formulated nucleic acid composition in reducing mRNA levels or protein levels.
  • Preparation of cationic amine stock solutions All organic amines and bis-amines are added to water to obtain a 100 millimolar solution and IN HCl was added dropwise until a pH of 7.1 is obtained. The resulting solution is filtered to 0.2 micron absolute using cell culture grade disposable filters prior to use. The solutions are stored at 5-8 C prior to use. All solutions remained free of precipitates during storage.
  • Additional instrumental techniques performed to characterize the cationic complexes include static light scattering and size exclusion chromatography using a Wyatt Technologies miniDawn detector with additional QELS hardware and Astra software (Wyatt Tecnologies, Santa Barbera, CA).
  • the size exclusion chromatography is performed using a TosoHaas TSK-gel SWxl column (4mm x 300mm) and Agilent 1 100 HPLC hardware including binary pump G1312A and PJD detector G1362A plus Chemstation software A.08.03.
  • This instrument can detect and quantitate hydrodynamic size of the cationic nucleic acid complexes and provide information on extinction coefficients of the nucleic acid component and molecular weight information of the complex through use of Zimm/ Rouse equations for light scattering.
  • a Brookhaven Instrument Corporation ZetaPALS dynamic light scattering instrument is used to measure size distributions of the cationic nucleic acid complexes and characterize the zeta-potential of these complexes.
  • Example 10 Formulation of polycationic complexes with proteins, peptides. or antibodies.
  • a protein, peptide, or antibody is complexed with a cationic compound based upon charge ratio or other properties such as hydrophobic interactions.
  • the complex can be formulated with different charge ratios by using equivalents of protein, peptide, or antibody to cation to generate a formulation with a net positive charge (e.g. excess cation to protein, peptide, or antibody), a neutral charge, or a net negative charge (e.g. excess protein, peptide, or antibody to cation).
  • the cation can be titrated into a solution of protein, peptide, or antibody or the protein, peptide, or antibody can be titrated into a solution of the cationic compound.
  • the protein, peptide, or antibody is obtained in HPLC purified form and dissolved in sterile Milli-Q water.
  • a stock solution of cationic amine is added in a suitable amount based upon the number of anionic charges present in the protein, peptide, or antibody.
  • an excess molar equivalence is used to generate a formulation with a net positive charge.
  • the resulting solution is analyzed by strong anion exchange chromatography for concentration and purity.
  • the solutions are then used in a cell culture assays to screen for efficacy of the formulated protein, peptide, or antibody composition in producing a therapeutic effect.
  • Analytical techniques including size exclusion analysis and light scattering analysis as described herein can be used to further characterize the formulations.
  • Example 11 Formulation of polycationic complexes with small molecules.
  • a small molecule e.g. small molecule drug
  • a cationic compound based upon charge ratio or other properties such as hydrophobic interactions.
  • the complex can be formulated with different charge ratios by using equivalents of small molecule to cation to generate a formulation with a net positive charge (e.g. excess cation to the small molecule), a neutral charge, or a net negative charge (e.g. excess small molecule to cation).
  • the cation can be titrated into a solution of the small molecule or the small molecule can be titrated into a solution of the cationic compound.
  • the small molecule is obtained in HPLC purified form and dissolved in sterile Milli-Q water.
  • a stock solution of cationic amine is added in a suitable amount based upon the number of anionic charges present in the small molecule. For example, to generate a formulation with a net positive charge, an excess molar equivalence is used. The resulting solution is analyzed by strong anion exchange chromatography for concentration and purity. The solutions are then used in a cell culture assays to screen for efficacy of the formulated small molecule composition in producing a therapeutic effect. Analytical techniques including size exclusion analysis and light scattering analysis as described herein can be used to further characterize the formulations.
  • formulated siNA molecules of the invention can be used to treat a variety of diseases and conditions through modulation of gene expression.
  • formulated siNA molecules can be designed to modulate the expression any number of target genes, including but not limited to genes associated with cancer, metabolic diseases, infectious diseases such as viral, bacterial or fungal infections, neurologic diseases, musculoskeletal diseases, diseases of the immune system, diseases associated with signaling pathways and cellular messengers, and diseases associated with transport systems including molecular pumps and channels.
  • Non-limiting examples of various viral genes that can be targeted using formulated siNA molecules of the invention include Hepatitis C Vims (HCV, for example Genbank Accession Nos: D11 168, D50483.1 , L38318 and S82227), Hepatitis B Virus (HBV, for example GenBank Accession No. AF 100308.1), Human Immunodeficiency Virus type 1 (HTV-1, for example GenBank Accession No. U51 188), Human Immunodeficiency Virus type 2 (HIV-2, for example GenBank Accession No. X60667), West Nile Virus (WNV for example GenBank accession No. NC_001563), cytomegalovirus (CMV for example GenBank Accession No.
  • HCV Hepatitis C Vims
  • HBV Hepatitis B Virus
  • HTV-1 Human Immunodeficiency Virus type 1
  • HTV-1 Human Immunodeficiency Virus type 1
  • HTV-1 Human Immunodeficiency
  • influenza virus for example GenBank Accession No. AF037412
  • rhinovirus for example, GenBank accession numbers: D00239, X02316, X01087, L24917, M16248, K02121, X01087
  • papillomavirus for example GenBank Accession No. NC 001353
  • Herpes Simplex Virus for example GenBank Accession No. NC 001345
  • other viruses such as HTLV (for example GenBank Accession No. AJ430458).
  • siNA molecules Due to the high sequence variability of many viral genomes, selection of siNA molecules for broad therapeutic applications would likely involve the conserved regions of the viral genome.
  • conserved regions of the viral genomes include but are not limited to 5 '-Non Coding Regions (NCR), 3'- Non Coding Regions (NCR) LTR regions and/or internal ribosome entry sites (IRES).
  • NCR Non-limiting examples of conserved regions of the viral genomes include but are not limited to 5 '-Non Coding Regions (NCR), 3'- Non Coding Regions (NCR) LTR regions and/or internal ribosome entry sites (IRES).
  • siNA molecules designed against conserved regions of various viral genomes will enable efficient inhibition of viral replication in diverse patient populations and may ensure the effectiveness of the siNA molecules against viral quasi species which evolve due to mutations in the non-conserved regions of the viral genome.
  • Non-limiting examples of human genes that can be targeted using formulated siNA molecules of the invention using methods described herein include any human RNA sequence, for example those commonly refened to by Genbank Accession Number. These RNA sequences can be used to design siNA molecules that inhibit gene expression and therefore abrogate diseases, conditions, or infections associated with expression of those genes.
  • Such non-limiting examples of human genes that can be targeted using siNA molecules of the invention include VEGFr (VEGFR 1 for example GenBank Accession No. XM 067723. VEGFR2 for example GenBank Accession No.
  • telomere for example GenBank Accession No. NM_003219
  • telomerase RNA for example GenBank Accession No. U86046
  • NFkappaB for example GenBank Accession No. NM_005228
  • NOGO for example GenBank Accession No. AB020693
  • NOGOr for example GenBank Accession No. XM 015620.
  • RAS for example GenBank Accession No. NM 004283).
  • RAF for example GenBank Accession No. XM 033884.
  • CD20 for example GenBank Accession No. X07203
  • METAP2 for example GenBank Accession No. NM_003219
  • CLCA1 for example GenBank Accession No. NM_001285
  • phospholamban for example GenBank Accession No. NM_002667
  • PTP1B for example GenBank Accession No. M31724
  • PCNA for example GenBank Accession No. NM_002592.1
  • PKC-alpha for example GenBank Accession No. NM_002737
  • the genes described herein are provided as non-limiting examples of genes that can be targeted using siNA molecules of the invention. Additional examples of such genes are described by accession number in Beigelman et al, USSN 60/363,124, filed March 11, 2002 and incorporated by reference herein in its entirety.
  • CAP any terminal cap, see for example Figure 10. All Stab 00-26 chemistries can comprise 3 '-terminal thymidine (TT) residues All Stab 00-26 chemistries typically comprise about 21 nucleotides, but can vary as described herein.
  • S sense strand

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Abstract

L'invention concerne l'administration de molécules actives sur le plan biologique dans des cellules. Plus précisément, l'invention concerne des compostions polycationiques, des polymères et des procédés permettant d'administrer des acides nucléiques, des polynucléotides et des oligonucléotides, tels que l'ARN, l'ADN et des analogues de ceux-ci, notamment le petit ARN interférent (ARNsi), des ribozymes et des antisens ou des peptides, des polypeptides, des protéines, des anticorps, des hormones et des petites molécules, dans des cellules et consistant à faciliter le transport dans les tissus épithéliaux et endothéliaux à membranes cellulaires. Les compositions et procédés selon l'invention sont utiles dans des applications thérapeutiques, de recherche et de diagnostic fondées sur le transfert efficace de molécules actives sur le plan biologique dans des cellules, des tissus et des organes.
EP04777833A 2003-07-09 2004-07-09 Compositions polycationiques destinees a l'administration cellulaire de polynucleotides Withdrawn EP1644500A2 (fr)

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US48566703P 2003-07-09 2003-07-09
US10/693,059 US20080039414A1 (en) 2002-02-20 2003-10-23 RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US10/720,448 US8273866B2 (en) 2002-02-20 2003-11-24 RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (SINA)
US10/757,803 US20050020525A1 (en) 2002-02-20 2004-01-14 RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
US10/826,966 US20050032733A1 (en) 2001-05-18 2004-04-16 RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (SiNA)
PCT/US2004/016390 WO2005019453A2 (fr) 2001-05-18 2004-05-24 Interference arn a mediation assuree par l'inhibition de genes au moyen de petit acide nucleique interferent (ansi) modifie chimiquement
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