Derivatized oligonucleotides having improved uptake and other properties

• US 7,235,650 B2
• Filed: 10/31/2002
• Issued: 06/26/2007
• Est. Priority Date: 01/11/1990
• Status: Expired due to Fees

• Current Assignee: ISIS Pharmaceuticals Incorporated
• Original Assignee: ISIS Pharmaceuticals Incorporated
• Inventors: Manoharan, Muthiah, Cook, Phillip Dan, Bennett, Clarence Frank
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US10/284,742
• Publication Number:
  US 20030175751A1
• Time in Patent Office: 1,699 Days
• Field of Search: 536/23.1, 536/25.3, 536/26.6, 435/6
• US Class Current: 536/23.1
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 47/542 Carboxylic acids, e.g. a fatty acid or an amino acid
• CPC Class Codes: A61K 47/551 one of the codrug's components being a vitamin, e.g. niacinamide, vitamin B3, cobalamin, vitamin B12, folate, vitamin A or retinoic acid
• CPC Class Codes: A61K 47/64 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 peptidic linkers A61K47/65
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07H 23/00 Compounds containing boron, silicon, or a metal, e.g. chelates, vitamin B12 esters with inorganic acids C07H11/00; metal salts, see parent compounds
• CPC Class Codes: C07J 43/003 not condensed
• CPC Class Codes: C07J 51/00 Normal steroids with unmodified cyclopenta(a)hydrophenanthrene skeleton not provided for in groups C07J1/00 - C07J43/00
• CPC Class Codes: C12N 15/113 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 when used in plants C12N15/8218
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• CPC Class Codes: C12N 2310/3125 Methylphosphonates
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/32 of the sugar
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/322 : 2'-R Modification
• CPC Class Codes: C12N 2310/33 : of the base
• CPC Class Codes: C12N 2310/333 : Modified A
• CPC Class Codes: C12N 2310/336 : Modified G
• CPC Class Codes: C12N 2310/3521 : Methyl
• CPC Class Codes: C12N 2310/3523 : Allyl
• CPC Class Codes: C12N 2310/3527 : Other alkyl chain
• CPC Class Codes: C12N 2310/3531 : Hydrogen
• CPC Class Codes: C12N 2310/3533 : Halogen
• CPC Class Codes: C12N 9/0069 : acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
• CPC Class Codes: C12Q 1/68 : involving nucleic acids
• CPC Class Codes: C12Y 113/11012 : Linoleate 13S-lipoxygenase (1.13.11.12)

Abstract

Linked nucleosides having at least one functionalized nucleoside that bears a substituent such as a steroid molecule, a reporter molecule, a non-aromatic lipophilic molecule, a reporter enzyme, a peptide, a protein, a water soluble vitamin, a lipid soluble vitamin, an RNA cleaving complex, a metal chelator, a porphyrin, an alkylator, a pyrene, a hybrid photonuclease/intercalator, or an aryl azide photo-crosslinking agent exhibit increased cellular uptake and other properties. The substituent can be attached at the 2′-position of the functionalized nucleoside via a linking group. If at least a portion of the remaining liked nucleosides are 2′-deoxy-2′-fluoro, 2′-O-methoxy, 2′-O-ethoxy, 2′-O-propoxy, 2′-O-aminoalkoxy or 2′-O-allyloxy nucleosides, the substituent can be attached via a linking group at any of the 3′ or the 5′ positions of the nucleoside or on the heterocyclic base of the nucleoside or on the inter-nucleotide linkage linking the nucleoside to an adjacent nucleoside.

Claims

1. A compound comprising a plurality of linked nucleosides including first and second functionalized nucleosides, wherein each of said first and second functionalized nucleosides is, independently:

a 2′

functionalized nucleoside having a reporter molecule linked to a 2′

-O-atom of the nucleoside;

a 5′

terminal nucleoside having a reporter molecule linked to a 5′

-O-atom of the nucleoside;

a 3′

terminal nucleoside having a reporter molecule linked to a 3′

-O-atom of the nucleoside;

or

an inter-strand nucleoside having a reporter molecule linked to an inter-nucleotide linkage linking said inter-strand nucleoside to an adjacent nucleoside

provided that the compound does not include sulfamate or sulfamide internucleotide linkages.

2. A compound of claim 1 wherein said first functionalized nucleoside comprises a 3′

terminal nucleoside having a reporter molecule linked to the 3′

-position of the nucleoside.

3. A compound of claim 1 wherein said second functionalized nucleoside comprises a 5′

terminal nucleoside having a reporter molecule linked to the 5′

-position of the nucleoside.

4. An oligonucleotide including at least first and second functionalized nucleosides, wherein each of said functionalized nucleosides is, independently:

a 2′

-functionalized nucleoside having a reporter molecule linked to a 2′

-O-atom of the nucleoside;

a 5′

terminal nucleoside having a reporter molecule linked to a 5′

-O-atom of the nucleoside;

a 3′

terminal nucleoside having a reporter molecule linked to a 3′

-O-atom of the nucleoside;

or

an inter-strand nucleoside having a reporter molecule linked to an inter-stand linkage linking said inter-strand nucleoside to an adjacent nucleoside

provided that the oligonucleotide does not include sulfamate or sulfamide internucleotide linkages.

5. The oligonucleotide of claim 4 wherein said first functionalized nucleoside is a 3′

terminal nucleoside having a reporter molecule linked to the 3′

-position of the nucleoside.

6. The oligonucleotide of claim 4 wherein said second functionalized nucleoside is a 5′

nucleoside having a reporter molecule linked to the 5′

-position of the nucleoside.

7. A method of enhancing an oligonucleotide for use in a research or diagnostic method comprising functionalizing said oligonucleotide with first and second reporter molecules, where at least one of said reporter molecules is linked to said oligonucleotide via a 2′

-O- or a 3′

-O atom of one of the nucleosides of said oligonucleotide and provided that the oligonucleotide does not include sulfamate or sulfamide internucletoide linkages.

8. The method of claim 7 wherein first and second reporter molecules each comprise reporter molecules linked to:

a 2′

-functionalized nucleoside having said reporter molecule linked to a 2′

-O-atom of a nucleoside of said oligonucleotide;

a 5′

terminal nucleoside having said reporter molecule linked to a 5′

-O-atom of a nucleoside of said oligonucleotide;

a 3′

terminal nucleoside having said reporter molecule linked to a 3′

-O-atom of a nucleoside of said oligonucleotide;

or

an inter-strand nucleoside of said oligonucleotide wherein said reporter molecule is linked to an inter-stand linkage linking said inter-strand nucleoside to an adjacent nucleoside of said oligonucleotide.

9. The method of claim 8 wherein said first reporter molecule is linked to a 3′

terminal nucleoside having said reporter molecule linked to the 3′

-position of the nucleoside.

10. The method of claim 8 wherein said second reporter molecule is linked to a 5′

nucleoside having said reporter molecule linked to the 5′

-position of the nucleoside.