Regulated aptamer therapeutics

• US 20060084109A1
• Filed: 11/30/2005
• Published: 04/20/2006
• Est. Priority Date: 07/25/2002
• Status: Active Grant

• Current Assignee: Charles Wilson, David Epstein, John L. Diener
• Original Assignee: Charles Wilson, David Epstein, John L. Diener
• Inventors: Epstein, David, Wilson, Charles, Diener, John L.
• Granted Patent: US 7,960,102 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12N 15/1048 SELEX
• CPC Class Codes: C12N 15/115 Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
• CPC Class Codes: C12N 2310/16 Aptamers
• CPC Class Codes: C12N 2310/3519 Fusion with another nucleic acid
• CPC Class Codes: C12N 2320/50 Methods for regulating/modulating their activity
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 2541/101 Selex
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: G01N 33/66 involving blood sugars, e.g. galactose

Abstract

Materials and methods of use thereof are presented for the treatment of diabetes and other diseases. Therapeutic compositions including regulated aptamer therapeutic compositions with specificity to components of diabetes disease are presented with methods of administering these therapeutic compositions.

Claims

1. A method of selecting a aptamer comprising the steps of:

a) identifying a first candidate aptamer comprising a first binding domain that binds to a first ligand and a second candidate aptamer comprising a second binding domain that binds to a second ligand;

b) engineering a candidate chimeric mixture wherein individual candidate oligonucleotides comprise a first and second binding domain of the first and second candidate aptamers;

c) and selecting, using SELEX™

, an aptamer from the candidate chimeric mixture wherein binding of the second ligand by the second binding domain of the selected aptamer is activated by binding of the first ligand by the first binding domain.

2. An aptamer obtainable by the method of claim 1.

3. The method of claim 1, wherein the identifying and engineering steps further comprise using SELEX™

to isolate a first nucleic acid mixture wherein individual candidate oligonucleotides comprise a first binding domain that binds to the first ligand and using the first nucleic acid mixture to engineer the candidate chimeric mixture.

4. The method of claim 3, wherein the identifying and engineering steps further comprise using SELEX™

to isolate a second nucleic acid mixture wherein individual candidate oligonucleotides comprise a second binding domain that binds to the second ligand and using the second nucleic acid mixture to engineer the candidate chimeric mixture.

5. The method of claim 4, wherein the selecting step further comprises contacting the chimeric candidate mixture with the second ligand, in a negative selection step, to result in a bound ligand and unbound ligand mixture and partitioning the unbound ligand mixture;

contacting only the unbound ligand mixture, in a positive selection step, with the first and second ligand to result in a second ligand bound mixture and unbound mixture; and

selecting an aptamer from the second ligand bound mixture

6. The method of claim 5, wherein the selecting step further comprises amplifying and contacting the second ligand bound mixture with the second ligand in the negative selection step and repeating the negative and positive selection steps iteratively prior to selecting the aptamer.

7. The method of claim 4, wherein the engineering step further comprises linearly concatenating the first and second ligand binding domains with an intervening random sequence domain.

8. The method of claim 4, wherein the engineering step further comprising coupling the first and second binding domains with linking helices.

18. The aptamer of claim 1, wherein the first ligand is an allosteric effector ligand.

19. The aptamer of claim 1, wherein the first ligand is a disease marker and the second ligand is a drug target.

20. The aptamer of claim 19, wherein the disease marker is glucose and the drug target is an insulin receptor.

9. A method of selecting an aptamer comprising the steps of:

a) identifying a first candidate aptamer comprising a first target binding domain that binds to a target;

b) linearly concatenating a random nucleic acid sequence domain to the target binding domain to result in a second nucleic acid mixture wherein individual candidate oligonucleotides comprise the target binding domain and a random nucleic acid sequence domain;

c) and selecting, using SELEX™

, an aptamer from the second nucleic acid mixture wherein binding of the target by the target binding domain of the aptamer is activated by binding of the aptamer to another ligand.

10. An aptamer obtainable by the method of claim 9.

11. The method of claim 9, wherein the identifying and concatenating steps further comprises using SELEX™

to isolate a target binding nucleic acid mixture wherein the individual candidate oligonucleotides comprise a target binding domain that binds to the target and linearly concatenating random nucleic acid sequence domains to target binding domains of the target binding nucleic acid mixture to result in the second nucleic acid mixture.

12. The method of claim 9, wherein the selecting step comprises contacting the second nucleic acid mixture, in a negative selection step, with the target to result in a bound target and unbound target mixture and partitioning the unbound target mixture;

contacting only the unbound target mixture with the target and ligand, in a positive selection step, to result in a second target bound and unbound mixture; and

selecting an aptamer from the second target bound mixture.

13. The method of claim 12, wherein the selecting step further comprises amplifying and contacting the second target bound mixture with the target in the negative selection step and repeating the negative and positive selection steps iteratively prior to selecting the aptamer.

14. The method of claim 9, wherein the selecting step comprises contacting the second nucleic acid mixture with the target, in a target enrichment step, to result in a target bound and unbound mixture, partitioning the unbound mixture, amplifying the target bound mixture to result in an enriched target bound mixture;

contacting the enriched target bound mixture, in a negative selection step, with the target to result in a bound target and unbound target mixture;

contacting only the unbound target mixture with the target and ligand, in a positive selection step, partitioning unbound mixture and amplifying target bound mixture; and

selecting the aptamer mixture from the amplified target bound mixture.

15. The method of claim 14, wherein the enrichment step comprises contacting the amplified, enriched target bound mixture with the target and repeating the enrichment step prior to the negative and positive selection steps.

16. The method of claim 15, wherein the selection step further comprises contacting the amplified target bound mixture with the target in the negative selection step and repeating the negative and positive selection steps iteratively prior to selecting the aptamer.

17. The method of claim 16, wherein sequences from the enriched target bound mixture are diversified prior to iterative repetition of the negative and positive selection steps.

21. A method for selecting a regulated aptamer which binds to a target and effector ligand, wherein the binding of the aptamer to the target is regulated by the binding of the aptamer to the effector ligand, comprising the following steps:

a) contacting a candidate nucleic acid mixture, wherein the individual oligonucleotides comprise a segment of randomized sequence, with a target to result in a target bound and unbound nucleic acid mixture and partitioning the unbound nucleic acid mixture;

b) contacting only the unbound nucleic acid mixture with the target and an effector ligand to result in a target bound and unbound mixture and amplifying the target bound mixture;

c) contacting the amplified target bound mixture with target and repeating steps a) and b) iteratively; and

d) selecting an aptamer that binds to the target in the presence of the effector ligand.

22. The method of claim 21, further comprising the steps of:

contacting a starting nucleic acid mixture, wherein the individual oligonucleotides comprise a segment of randomized sequence, with the target to result in a target bound and unbound mixture, partitioning the unbound mixture and amplifying the target bound mixture;

contacting the amplified target bound mixture with the target, partitioning the unbound mixture and amplifying the target bound mixture and then contacting the enriched target bound nucleic acid mixture with the target in step a).

23. The method of claim 21, further comprising the steps of:

contacting a starting nucleic acid mixture, wherein the individual oligonucleotides comprise a segment of randomized sequence, with the effector ligand to result in a effector ligand bound and unbound mixture, partitioning the unbound mixture and amplifying the effector ligand bound mixture;

contacting the amplified effector ligand bound mixture with the effector ligand, partitioning the unbound mixture and amplifying the effector ligand bound mixture and then contacting the amplified, enriched effector ligand bound nucleic acid mixture with the target in step a).

24. An aptamer obtainable by the method of claim 21.