Methods for the Delivery of Oligomeric Compounds

• US 20080199960A1
• Filed: 05/13/2005
• Published: 08/21/2008
• Est. Priority Date: 05/13/2004
• Status: Abandoned Application

• Current Assignee: KU Leuven Research & Development, University of North Carolina At Chapel Hill (University of North Carolina System)
• Original Assignee: KU Leuven Research & Development
• Inventors: Herdewijn, Piet, Van Aerschot, Arthur, Juliano, Rudolph L., Chaltin, Patrick, Kang, Hyunmin, Astriab-Fisher, Anna
• Application Number:
  US11/596,238
• Publication Number:
  US 20080199960A1
• Time in Patent Office: Days
• US Class Current: 435/440
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/1138 against receptors or cell surface proteins
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/322 2'-R Modification
• CPC Class Codes: C12N 2310/3231 having an additional ring, e.g. LNA, ENA
• CPC Class Codes: C12N 2310/341 Gapmers, i.e. of the type ===---===
• CPC Class Codes: C12N 2310/3513 Protein; Peptide
• CPC Class Codes: C12N 2310/3515 Lipophilic moiety, e.g. cholesterol
• CPC Class Codes: C12N 2310/3521 Methyl
• CPC Class Codes: C12N 2310/3525 MOE, methoxyethoxy
• CPC Class Codes: C12N 2310/3527 Other alkyl chain
• CPC Class Codes: C12N 2310/53 partially self-complementary or closed
• CPC Class Codes: C12N 2320/32 Special delivery means, e.g. tissue-specific
• CPC Class Codes: C12N 2320/51 modulating the chemical stability, e.g. nuclease-resistance

Abstract

The presently disclosed subject matter relates to the delivery of oligonucleotides to cells through the delivery of a composition or reagent comprising a hybridization complex comprising a first antisense oligonucleotide which is modified to have a higher stability against degradation, and a second sense oligonucleotide which is prone to degradation. The presently disclosed subject matter furthermore relates to dendrimeric bioconjugates and compositions or reagents comprising them, wherein the bioconjugate comprises a conjugate moiety coupled to a dendrimeric structure and to their use to deliver oligomeric compounds including oligonucleotides or duplexes, as described above, to cells for modulation of gene expression (i.e. antisense or antigene therapy/research, RNA interference).

Claims

1. A method of inhibiting the expression of a gene in a cell, the method comprising administering to the cell a composition comprising a hybridization complex, wherein the hybridization complex comprises:

(a) a first oligonucleotide strand, wherein the first oligonucleotide strand comprises a fully or partially modified oligonucleotide that hybridizes to at least a portion of an mRNA molecule transcribed from the gene; and

(b) a second oligonucleotide strand, wherein the second oligonucleotide strand comprises a deoxyribonucleic acid that is complementary to at least a portion of the first strand, whereby the first oligonucleotide strand and the second oligonucleotide strand enter the cell and the expression of the gene in the cell is inhibited.

2. The method of claim 1, wherein the fully or partially modified oligonucleotide comprises a modification selected from the group consisting of a internucleoside linkage modification or carbohydrate modification.

3. The method of claim 2, wherein the internucleoside linkage modification comprises one or more phosphorothioate linkages.

4. The method of claim 2, wherein the carbohydrate modification comprises a 2′

modification.

5. The method of claim 4, wherein the 2′

modification is selected from the group consisting of 2′

-halo, 2′

-O-methyl, 2′

-O-ethyl, 2′

-O-propyl, 2′

-O-butyl;

2′

-O-pentyl, 2′

-O-(2-methoxyethyl), and 2′

-O-[2-[N,N-dimethylamino)oxy]-ethyl].

6. The method of claim 4, wherein the 2′

modification comprises a locked nucleic acid, wherein the locked nucleic acid is characterized by a methylene bridge that connects a 2′

-oxygen with a 4′

-carbon of a ribose.

7. The method of claim 2, wherein the first oligonucleotide strand has the following general structure:

(2′

-O-modified ribonucleotide)_x-(deoxyribonucleotide)_y-(2′

-O-modified ribonucleotide)_z.

wherein y is at least 5 and x+y+z equals at least 18 but less than 30.

8. The method of claim 7, wherein when y equals 5, x and z differ by no more than 2.

9. The method of claim 7, wherein y is at least 7.

10. The method of claim 7, wherein the 2′

-O-modified ribonucleotide comprises a 2′

-O-methyl group.

11. The method of claim 1, wherein the second oligonucleotide strand is shorter than or equal in length to the first oligonucleotide strand.

12. The method of claim 1, wherein the composition further comprises a cationic lipid.

13. The method of claim 12, wherein the composition further comprises a bioconjugate dendrimer, wherein the bioconjugate dendrimer comprises a conjugate moiety coupled to a dendrimeric structure.

14. The method of claim 1, wherein the second oligonucleotide strand is an unmodified deoxyribonucleic acid.

15. The method of claim 1, wherein the second oligonucleotide strand is modified at the 5′

-end or the 3′

-end to a conjugate moiety.

16. The method of claim 15, wherein the modification comprises the conjugation of a conjugate moiety selected from the group of a cholesterol moiety or a peptide.

17. The method of claim 15, wherein the modification comprises the coupling of the second oligonucleotide to one or more other oligonucelotides and thereby forming a dendrimeric structure, which is further conjugated to a conjugate moiety.

18. A method of enhancing the uptake of a single-stranded oligonucleotide by a cell, the method comprising:

(a) hybridizing to the single-stranded oligonucleotide a deoxyribonucleic acid molecule that is complementary to a subsequence of the single-stranded oligonucleotide to create a double-stranded molecule; and

(b) contacting the cell with the double-stranded molecule, whereby uptake of the single-stranded oligonucleotide by the cell is enhanced.

19. The method of claim 18, wherein the single-stranded oligonucleotide comprises a modification selected from the group consisting of a internucleoside linkage modification and a carbohydrate modification.

20. The method of claim 19, wherein the internucleoside linkage modification comprises one or more phosphorothioate linkages.

21. The method of claim 18, wherein the carbohydrate modification comprises a 2′

modification.

22. The method of claim 21, wherein the 2′

modification is selected from the group consisting of 2′

-halo, 2′

-O-methyl, 2′

-O-ethyl, 2′

-O-propyl, 2′

-O-butyl;

2′

-O-pentyl, 2′

-O-(2-methoxyethyl), and 2′

-O-[2-[N,N-dimethylamino)oxy]-ethyl].

23. The method of claim 21, wherein the 2′

modification comprises a locked nucleic acid, wherein the locked nucleic acid is characterized by a methylene bridge that connects a 2′

-oxygen with a 4′

-carbon of a ribose.

24. The method of claim 18, wherein the single-stranded oligonucleotide has the following general structure:

(2′

-O-modified ribonucleotide)_x-(deoxyribonucleotide)_y-(2′

-O-modified ribonucleotide)_z, wherein y is at least 5 and x+y+z equals at least 18 but less than 30.

25. The method of claim 24, wherein when y equals 5, x and z differ by no more than 2.

26. The method of claim 24, wherein y is at least 7.

27. The method of claim 24, wherein the 2′

-O-modified ribonucleotide comprises a 2′

-O-methyl group.

28. The method of claim 18, wherein the deoxyribonucleic acid molecule is shorter than or equal in length to the single-stranded oligonucleotide.

29. The method of claim 18, wherein the method further comprises the addition of a cationic lipid.

30. The method of claim 29, wherein the methode further comprises the addition of a bioconjugate dendrimer, wherein the bioconjugate dendrimer comprises a conjugate moiety coupled to a dendrimeric structure.

31. The method of claim 18, wherein the deoxyribonucleic acid molecule comprises an unmodified deoxyribonucleic acid.

32. The method of claim 18, wherein the deoxyribonucleic acid molecule further comprises a modification selected from the group consisting of a 5′

-end modification and a 3′

-end modification.

33. The method of claim 32, wherein the modification comprises a conjugated cholesterol moiety or a conjugated peptide.

34. A composition comprising a dendrimeric bioconjugate and an oligomeric compound, wherein the dendrimeric bioconjugate comprises a conjugate moiety coupled to a dendrimeric structure.

35. The composition of claim 34, wherein the dendrimeric structure is selected from the group of a cationic polymer such as PAMAM and a dendrimeric oligonucleotide structure.

36. The composition of claim 34, wherein the conjugate moiety is selected from the group of lipidic molecules such as cholesterol and peptides.

37. The composition of claim 34, wherein the oligomeric compound comprises a hybridization complex, and wherein the hybridization complex comprises:

(i) a first oligonucleotide strand, wherein the first oligonucleotide strand comprises a fully or partially modified oligonucleotide that hybridizes to at least a portion of an mRNA molecule transcribed from the gene; and

(ii) a second oligonucleotide strand, wherein the second oligonucleotide strand comprises a deoxyribonucleic acid that is complementary to at least a portion of the first strand.

38. The composition of claim 34, further comprising a cationic lipid.

39. A method of enhancing the uptake of an oligomeric compound, the method comprising:

(a) combining the oligomeric compound with a dendrimeric bioconjugate in a composition, wherein the dendrimeric bioconjugate comprises a conjugate moiety coupled to a dendrimeric structure; and

(b) contacting the cell with the composition.

40. A method of inhibiting the expression of a gene in a cell, the method comprising administering to the cell a composition comprising an oligomeric compound and a dendrimeric bioconjugate, wherein the dendrimeric bioconjugate comprises a conjugate moiety coupled to a dendrimeric structure.

41. The method of claim 40, wherein the dendrimeric structure is selected from the group of a cationic polymer such as PAMAM and a dendrimeric oligonucleotide structure.

42. The method of claim 40, wherein the conjugate moiety is selected from the group of lipidic molecules such as cholesterol and peptides.

43. A method for the preparation of cholesterol-dendrimeric oligonucleotide bioconjugate, the method comprising:

(a) derivatizing cholesterol to obtain a free carboxylgroup;

(b) coupling to the derivatized cholesterol Fmoc-lysine-OMe or an analogous structure comprising a protected amino group and a protected carboxylgroup which are separately cleavable;

(c) deprotecting the carboxyl group;

(d) coupling the obtained compound to a solid phase through an acid labile function, like an ester;

(e) deprotecting the amino group;

(f) repetitive steps of

[1] coupling of a compound with two protected amino functions and a free carboxylgroup like (Fmoc)₂-Lys-OH; and

[2] deprotecting the amino-funtion protecting groups according to the amount of dendrimeric units requested;

(g) coupling a linker with a free hydroxyl as reactive group; and

(h) performing standard oligonucleotide synthesis followed by cleaving the compound of from the solid phase.

44. A method of enhancing the uptake in a cell of an oligomeric compound mediating RNA interference such as siRNAs, the method comprising combining the oligomeric compound with a dendrimeric structure such as a cationic polymer.