Human type 2 RNase H

• US 6,001,653 A
• Filed: 12/02/1998
• Issued: 12/14/1999
• Est. Priority Date: 12/04/1997
• Status: Expired due to Term

• Current Assignee: Ionis Pharmaceuticals, Inc.
• Original Assignee: ISIS Pharmaceuticals Incorporated
• Inventors: Crooke, Stanley T., Lima, Walter F., Wu, Hongjiang
• Primary Examiner(s): LeGuyader, John L.
• Application Number:
  US09/203,716
• Time in Patent Office: 377 Days
• Field of Search: 435/6, 435/91.1, 435/320.1, 435/375, 435/440, 435/325, 435/455, 435/366, 435/193, 536/23.1, 536/23.2, 536/23.5, 536/24.3, 536/24.31, 536/24.33, 536/24.5, 536/25.3
• US Class Current: 435/375
• 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: 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 2310/11 Antisense
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

The present invention provides polynucleotides and polypeptides encoded thereby of human Type 2 RNase H. Methods of using these polynucleotides and polypeptides in enhancing antisense oligonucleotide therapies are also provided.

Claims

1. An isolated polynucleotide encoding the human Type 2 RNase H polypeptide (SEQ ID NO:

1).

2. A vector comprising a nucleic acid encoding the human Type 2 RNase H polypeptide (SEQ ID NO:

1).

3. A host cell comprising the vector of claim 2.

4. A composition comprising a vector comprising a nucleic acid encoding the human Type 2 RNase H polypeptide (SEQ ID NO:

1).

5. The composition of claim 4 further comprising an antisense oligonucleotide.

6. A nucleic acid probe which hybridizes to a nucleic acid encoding the human Type 2 RNase H polypeptide (SEQ ID NO:

1).

7. An antisense oligonucleotide which cleaves its complementary target RNA by the human RNase H Type 2 polypeptide (SEQ ID NO:

1), wherein the antisense oligonucleotide is a chimeric oligonucleotide with 2'"'"' methoxy flanks and a 5 deoxynucleotide center gap.

8. A composition comprising an antisense oligonucleotide and a human Type 2 RNase H polypeptide, wherein the human Type 2 RNase H polypeptide is the human RNase H Type 2 polypeptide (SEQ ID NO:

1).

9. A human Type 2 RNase H-his-tag fusion polypeptide, wherein the human Type 2 RNase H polypeptide is the human RNase H Type 2 polypeptide (SEQ ID NO:

1).

10. A method of enhancing inhibition of expression of a selected protein by an antisense oligonucleotide targeted to an RNA encoding the selected protein comprising, targeting the antisense oligonucleotide, which antisense oligonucleotide is a chimeric oligonucleotide with 2'"'"' methoxy flanks and a 5 deoxynucleotide center gap, to the RNA such that the antisense oligonucleotide hybridizes to form an antisense oligonucleotide-RNA duplex, wherein the antisense oligonucleotide-RNA duplex is cleaved by the human Type 2 RNase H polypeptide (SEQ ID NO:

1), whereby inhibition of expression of the selected protein is enhanced.

11. A method of making an antisense oligonucleotide which is cleaved by the human Type 2 RNase H polypeptide (SEQ ID NO:

1) comprising synthesizing an oligonucleotide which is targeted to a selected RNA wherein the antisense oligonucleotide, when hybridized to the selected RNA target to from a duplex, will bind the human Type 2 RNase H polypeptide and thereby cleave the duplex.

12. A method of screening oligonucleotides to identify effective antisense oligonucleotides for inhibition of expression of a selected target protein comprising:

(a) contacting the human Type 2 RNase H polypeptide (SEQ ID NO;

     1) with an RNA encoding the selected target protein and an oligonucleotide complementary to at least a portion of the RNA under conditions in which an oligonucleotide-RNA duplex is formed;

(b) detecting cleavage of the RNA of the oligonucleotide-RNA duplex wherein cleavage is indicative of antisense efficacy.

13. The method of claim 12 further comprising determining the site on the RNA at which cleavage occurs, whereby said site is identified as a Type 2 RNase H-sensitive site.

14. The method of claim 13 further comprising identifying an effective antisense oligonucleotide which hybridizes to said Type 2 RNase H-sensitive site.

15. The method of claim 12 wherein the oligonucleotide is one of a mixture or library of oligonucleotides.

16. An antisense oligonucleotide obtained by the method of claim 14.

17. A method of identifying agents which increase or decrease activity or levels of the human Type 2 RNase H polypeptide (SEQ ID NO:

1) in a host cell comprising;

(a) contacting a cell in vitro expressing the human type II RNase H polypeptide with an agent suspected or increasing or decreasing activity or levels of the human RNase H polypeptide; and

(b) measuring the activity or levels of the human RNase H polypeptide in the presence and absence of the agent so that an increase or decrease in the activity or levels of the human RNase H polypeptide can be determined.