Compositions and Methods for Modulating Gene Expression Using Self-Protected Oligonucleotides

• US 20090005332A1
• Filed: 12/30/2005
• Published: 01/01/2009
• Est. Priority Date: 12/30/2004
• Status: Abandoned Application

• Current Assignee: Aaron Loomis, Todd M. Hauser
• Original Assignee: Aaron Loomis, Todd M. Hauser
• Inventors: Hauser, Todd M., Loomis, Aaron
• Application Number:
  US11/813,190
• Publication Number:
  US 20090005332A1
• Time in Patent Office: Days
• US Class Current: 514/44.R
• CPC Class Codes: A61P 19/04 for non-specific disorders of the connective tissue
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/10 Antimycotics
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 33/02 Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/1135 against oncogenes or tumor suppressor genes
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/111 spanning the whole gene, or a large part of it
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/53 partially self-complementary or closed
• CPC Class Codes: C12N 2320/51 modulating the chemical stability, e.g. nuclease-resistance

Abstract

The present invention is directed to novel nucleic acid molecules which include a region complementary to a target gene and one or more self-complementary regions, and the use of such nucleic acid molecules and compositions comprising the same to modulate gene expression and treat a variety of diseases and infections.

Claims

1. An isolated polynucleotide comprising a region having a sequence complementary to a target gene or mRNA sequence and one or more self-complementary regions.

2. The polynucleotide of claim 1, wherein said polynucleotide comprises two or more self-complementary regions.

3. The polynucleotide of claim 1, wherein said polynucleotide comprises RNA.

4. The polynucleotide of claim 1, wherein said polynucleotide comprises DNA.

5. The polynucleotide of claim 1, wherein said polynucleotide comprises a peptide nucleic acid.

6. The polynucleotide of claim 1, wherein said self-complementary regions are located at the 5′

or 3′

or both ends of the polynucleotide.

7. The polynucleotide of claim 1, further comprising one or more additional regions of sequence complementary to a target gene or mRNA sequence, wherein said regions of sequence complementary to a target gene or mRNA sequence are separated by self-complementary regions are located at the 5′

or 3′

or both ends of the polynucleotide.

8. The polynucleotide of claim 7, wherein said regions of sequence complementary to a target gene or mRNA sequence are complementary to the same target gene or mRNA sequence.

9. The polynucleotide of claim 7, wherein said regions of sequence complementary to a target gene or mRNA sequence are complementary to two or more different genes or mRNA sequences.

10. The polynucleotide of claim 1, further comprising a second sequence that is non-complementary or semi-complementary to a target gene or mRNA sequence and non-complementary to a self-complementary region, wherein said second sequence is located between the self-complementary region and the sequence complementary to a target gene or mRNA sequence.

11. The polynucleotide of claim 1, wherein said self-complementary region comprises a stem-loop structure.

12. The polynucleotide of claim 1, wherein said self-complementary region does not complement the sequence complementary to a target gene or mRNA sequence.

13. The polynucleotide of claim 1, wherein said polynucleotide comprises two self-complementary regions, and wherein said two self-complementary regions do not complement each other.

14. The polynucleotide of claim 1, wherein said sequence complementary to a target gene or mRNA sequence comprises at least 17 nucleotides.

15. The polynucleotide of claim 14, wherein said sequence complementary to a target gene or mRNA sequence comprises 17 to 30 nucleotides.

16. The polynucleotide of claim 14, wherein said self-complementary region comprises at least 5 nucleotides.

17. The polynucleotide of claim 14, wherein said self-complementary region comprises at least 24 nucleotides.

18. The polynucleotide of claim 14, wherein said self-complementary region comprises 12 to 48 nucleotides.

19. The polynucleotide of claim 11, wherein said loop comprises at least 4 nucleotides.

20. An array comprising a plurality of polynucleotides of claim 1.

21. An expression vector encoding a polynucleotide of claim 1.

22. A composition comprising a physiologically acceptable carrier and a polynucleotide of claim 1.

23. A method for reducing the expression of a gene, comprising introducing an isolated polynucleotide of claim 1 into a cell.

24. The method of claim 23, wherein the cell is plant, animal, protozoan, viral, bacterial, or fungal.

25. The method of claim 23, wherein the cell is mammalian.

26. The method of claim 23, wherein the isolated polynucleotide is introduced directly into the cell.

27. The method of claim 23, wherein the isolated polynucleotide is introduced extracellularly by a means sufficient to deliver the isolated polynucleotide into the cell.

28. A method for treating a disease, comprising introducing an isolated polynucleotide of claim 1 into a cell, wherein expression of the gene or mRNA is associated with the disease.

29. The method of claim 28, wherein the disease is a cancer.

30. A method of treating an infection in a patient, comprising introducing into the patient the isolated polynucleotide of claim 1, wherein the isolated polynucleotide mediates entry, replication, integration, transmission, or maintenance of an infective agent.

31. A method for identifying a function of a gene, comprising:

(a) introducing into a cell the isolated polynucleotide of claim 1, wherein the isolated polynucleotide inhibits expression of the gene; and

(b) determining the effect of step (a) on a characteristic of the cell, thereby determining the function of the gene.

32. The method of claim 31, wherein the method is performed using high throughput screening.

34. The polynucleotide of claim 1, wherein said polynucleotide exhibits an increased half-life in vivo, as compared to the same polynucleotide lacking the one or more self-complementary regions.

35. A method for treating a disease, comprising introducing an isolated polynucleotide of claim 1 into a cell, wherein said gene or mRNA comprises one or more mutations as compared to a corresponding wild-type gene or mRNA.

36. The method of claim 35, wherein said disease is cystic fibrosis.

37. A method of modulating the expression of a mutated gene or mRNA in a cell, comprising introducing a polynucleotide of claim 1 into a cell, wherein said target gene or mRNA sequence comprises a region of said mutated gene or mRNA.

38. The method of claim 37, wherein said mutated gene or mRNA is associated with cystic fibrosis.

39. The method of claim 38, wherein said mutated mRNA is an mRNA expressed from a gene encoding a mutant Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) polypeptide.

40. The method of claim 37, wherein said mutated gene or mRNA is associated with a tumor.

41. The method of claim 40, wherein said mutated mRNA is an mRNA expressed from a gene encoding a mutant p53 polypeptide.

42. The method of claim 23, wherein said target gene is a transactivator that drives the expression of a second gene.

43. The method of claim 23, wherein said target gene is a repressor that inhibits expression of a second gene.

33. A method of designing a polynucleotide sequence comprising one or more self-complementary regions for the regulation of expression of a target gene or mRNA, comprising:

(a) selecting a first sequence 17 to 30 nucleotides in length and complementary to a target gene or mRNA;

(b) selecting one or more additional sequences 12 to 48 nucleotides in length, which comprises self-complementary regions and which are non-complementary to the first sequence; and

(c) selecting one or more further additional sequences 2 to 12 nucleotides in length, which are non-complementary or self-complementary to the target gene or mRNA and which are non-complementary to the additional sequences selected in step (b),

thereby designing a polynucleotide sequence for the regulation of expression of a target gene or mRNA.