Regulatable RNA molecule

• US 5,834,186 A
• Filed: 06/02/1995
• Issued: 11/10/1998
• Est. Priority Date: 12/04/1992
• Status: Expired due to Fees

• Current Assignee: Sirna Therapeutics, Inc. (Alnylam Pharmaceuticals Incorporated)
• Original Assignee: Innovir Laboratories, Inc. (Baxter International Inc.)
• Inventors: George, Shaji T., Shih, Andy, Bockman, Jeffrey Michael
• Primary Examiner(s): LeGuyader, John L.
• Application Number:
  US08/460,194
• Time in Patent Office: 1,257 Days
• Field of Search: 435/6, 435/91.1, 435/91.31, 435/172.1, 435/172.3, 435/320.1, 435/240.1, 435/240.2, 435/240.4, 435/252.3, 536/23.1, 536/23.2, 536/24.5
• US Class Current: 435/5
• 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: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 31/20 for DNA viruses
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• 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 2310/121 Hammerhead
• CPC Class Codes: C12N 2730/10122 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Abstract

Regulatable RNA molecules such as regulatable ribozymes, nucleic acids encoding such regulatable ribozymes, and methods of making and using such regulatable ribozymes are disclosed. Regulatable ribozymes comprise a ligand-binding RNA sequence and a ribozyme sequence capable of cleaving a separate targeted RNA sequence, wherein upon binding of the ligand to the ligand-binding RNA sequence, the activity of the ribozyme sequence against the targeted RNA sequence is altered. The ligand may be either an inorganic or an organic molecule and may be a co-drug which can be administered to specifically regulate the ribozyme activity. Regulatable RNA molecules other than ribozymes are also disclosed, such as regulatable mRNA molecules which comprise a ligand-binding RNA sequence separate from the coding sequence, wherein upon binding of a ligand to the ligand-binding RNA sequence, translation of the regulatable mRNA is altered.

Claims

1. An RNA molecule comprising a first RNA sequence having a biological function and a ligand-binding second RNA sequence, wherein the binding of the ligand to the ligand-binding second RNA sequence alters the biological function of the first RNA sequence.

2. The RNA molecule of claim 1 wherein the first RNA sequence is an mRNA sequence expressed in cells.

3. The RNA molecule of claim 1 wherein the ligand is selected from the group consisting of nucleic acid molecules, proteins, polysaccharides, sugars, and organic and inorganic molecules.

4. The RNA molecule of claim 1 wherein the first RNA sequence is an external guide sequence for RNAase P.

5. The RNA molecule of claim 1 wherein the biological function of the first RNA sequence is inactivated when the ligand is bound to the molecule.

6. The RNA molecule of claim 1 wherein the biological function of the first RNA sequence is activated when the ligand is bound to the molecule.

7. The RNA molecule of claim 3 wherein ligand is a product found in cells and the biological function of the first RNA sequence is altered by binding of the RNA molecule to the ligand within the cells.

8. The RNA molecule of claim 1, wherein the RNA molecule is made by

providing a random nucleotide sequence which is linked to the first RNA sequence having a biological function,

amplifying the random nucleotide sequence under conditions promoting mutation of the random sequence,

exposing the mutated random sequence to the ligand which is to be used to alter the biological function of the first RNA sequence under conditions favorable to binding of the mutated nucleotide sequence to the ligand,

removing mutated random nucleotide sequence not binding to the ligand,

repeating the amplification and binding steps until the mutated second RNA sequence is obtained which binds to the ligand,

exposing the RNA molecule to ligand, and

screening for alteration of the biological activity of the first RNA sequence when ligand is bound and when ligand is not bound to the ligand-binding mutated second RNA sequence.

9. A method of making a regulatable RNA molecule comprising a first RNA sequence having a biological function and a ligand-binding mutated second RNA sequence, wherein binding of the ligand to the mutated second RNA sequence alters the biological activity of the first RNA sequence,

comprising the steps of:

providing a random nucleotide sequence which is linked to the first RNA sequence having a biological function,

amplifying the random nucleotide sequence under conditions promoting mutation of the random sequence,

exposing the mutated random sequence to the ligand which is to be used to alter the biological function of the first RNA sequence under conditions favorable to binding of the mutated second RNA sequence to the ligand,

removing mutated random nucleotide sequence not binding to the ligand,

repeating the amplification and binding steps until the mutated second RNA sequence is obtained which binds to the ligand,

exposing the RNA molecule to ligand, and

screening for alteration of the biological activity of the first RNA sequence when ligand is bound and when ligand is not bound to the ligand-binding mutated second RNA sequence.

10. The method according to claim 9 wherein the first RNA sequence is an mRNA sequence expressed in cells.

11. The method of claim 9 wherein the ligand is selected from the group consisting of nucleic acid molecules, proteins, polysaccharides, sugars, and organic and inorganic molecules.

12. The method of claim 9 wherein the first RNA sequence is an external guide sequence for RNAase P.

13. The method of claim 9 wherein the biological activity of the first RNA sequence is inactivated when the ligand is bound to the RNA molecule.

14. The method of claim 9 wherein the biological activity of the first RNA sequence is activated when the ligand is bound to the RNA molecule.

15. The method of claim 9 wherein ligand is a product found in cells and the biological activity of the first RNA sequence is altered by binding to the ligand within the cells.

16. The method of claim 9 wherein ligand is a product found in virally infected cells and the biological activity of the first RNA sequence is activated by binding to the ligand.