IP Library › Granted Patent US 7,355,035
Granted Patent B1
US 7,355,035 · App. 09/889,075 · Granted Apr 8, 2008

Catalytic molecules

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Quick Facts
Patent No.
US 7,355,035
App. No.
09/889,075
Granted
Apr 8, 2008
Kind
B1
Abstract

The present invention relates to DNAzymes which are targeted against mRNA molecules encoding EGR-1 (also known as Egr-1 and NGFI-A). The present invention also relates to compositions including these DNAzymes and to methods of treatment involving administration of the DNAzymes.

Claims (70)

1. A DNAzyme which specifically cleaves EGR-1 mRNA, the DNAzyme comprising

(i) a catalytic domain which cleaves mRNA at a purine:pyrimidine cleavage site;

(ii) a first binding domain continuous with the 5′ end of the catalytic domain; and

(iii) a second binding domain continuous with the 3′ end of the catalytic domain,

wherein the binding domains are sufficiently complementary to the two regions immediately flanking a purine:pyrimidine cleavage site within the region of EGR-1 mRNA corresponding to nucleotides 168-332 as shown in SEQ ID No: 1, such that the DNAzyme cleaves the EGR-1 mRNA.

2. A DNAzyme as claimed in claim 1 wherein the 3′-end nucleotide residue is inverted in the binding domain contiguous with the 3′-end of the catalytic domain.

3. A DNAzyme as claimed in claim 1 in which the cleavage site is selected from the group consisting of

(i) the GU site corresponding to nucleotides 198-199;

(ii) the GU site corresponding to nucleotides 200-201;

(iii) the GU site corresponding to nucleotides 264-265;

(iv) the AU site corresponding to nucleotides 271-272;

(v) the AU site corresponding to nucleotides 301-302;

(vi) the GU site corresponding to nucleotides 303-304; and

(vii) the AU site corresponding to nucleotides 316-317.

4. A DNAzyme as claimed in claim 3 in which the cleavage site is the AU site corresponding to nucleotides 271-272.

5. A DNAzyme as claimed in claim 3 wherein the 3′-end nucleotide residue is inverted in the binding domain contiguous with the 3′-end of the catalytic domain.

6. A DNAzyme as claimed in claim 4 wherein the 3′-end nucleotide residue is inverted in the binding domain contiguous with the 31-end of the catalytic domain.

7. A DNAzyme as claimed in claim 1 in which the catalytic domain has the nucleotide sequence GGCTAGCTACAACGA [SEQ. ID. NO:2].

8. A DNAzyme as claimed in claim 7 wherein the 3′-end nucleotide residue is inverted in the binding domain contiguous with the 3′-end of the catalytic domain.

9. A DNAzyme as claimed in claim 7 in which the cleavage site is selected from the group consisting of

(i) the GU site corresponding to nucleotides 198-199;

(ii) the GU site corresponding to nucleotides 200-201;

(iii) the GU site corresponding to nucleotides 264-265;

(iv) the AU site corresponding to nucleotides 271-272;

(v) the AU site corresponding to nucleotides 301-302;

(vi) the GU site corresponding to nucleotides 303-304; and

(vii) the AU site corresponding to nucleotides 316-317.

10. A DNAzyme as claimed in claim 9 in which the cleavage site is the AU site corresponding to nucleotides 271-272.

11. A DNAzyme as claimed in claim 9 wherein the 3′-end nucleotide residue is inverted in the binding domain contiguous with the 3′-end of the catalytic domain.

12. A DNAzyme as claimed in claim 9 wherein the 3′-end nucleotide residue is inverted in the binding domain contiguous with the 3′-end of the catalytic domain.

13. A DNAzyme as claimed in claim 1 wherein each binding domain is nine or more nucleotides in length.

14. A DNAzyme as claimed in claim 13 wherein the 3′-end nucleotide residue is inverted in the binding domain contiguous with the 3′-end of the catalytic domain.

15. A DNAzyme as claimed in claim 13 in which the cleavage site is selected from the group consisting of

(i) the GU site corresponding to nucleotides 198-199;

(ii) the GU site corresponding to nucleotides 200-201;

(iii) the GU site corresponding to nucleotides 264-265;

(iv) the AU site corresponding to nucleotides 271-272;

(v) the AU site corresponding to nucleotides 301-302;

(vi) the GU site corresponding to nucleotides 303-304; and

(vii) the AU site corresponding to nucleotides 316-317.

16. A DNAzyme as claimed in claim 15 in which the cleavage site is the AU site corresponding to nucleotides 271-272.

17. A DNAzyme as claimed in claim 15 wherein the 3′-end nucleotide residue is inverted in the binding domain contiguous with the 3′-end of the catalytic domain.

18. A DNAzyme as claimed in claim 16 wherein the 3′-end nucleotide residue is inverted in the binding domain contiguous with the 3′-end of the catalytic domain.

19. A DNAzyme as claimed in claim 13 in which the catalytic domain has the nucleotide sequence GGCTAGCTACAACGA [SEQ ID NO: 2].

20. A DNAzyme as claimed in claim 19 wherein the 3′-end nucleotide residue is inverted in the binding domain contiguous with the 3′-end of the catalytic domain.

21. A DNAzyme as claimed in claim 19 in which the cleavage site is selected from the group consisting of

(i) the GU site corresponding to nucleotides 198-199;

(ii) the GU site corresponding to nucleotides 200-201;

(iii) the GU site corresponding to nucleotides 264-265;

(iv) the AU site corresponding to nucleotides 271-272;

(v) the AU site corresponding to nucleotides 301-302;

(vi) the GU site corresponding to nucleotides 303-304; and

(vii) the AU site corresponding to nucleotides 316-317.

22. A DNAzyme as claimed in claim 21 in which the cleavage site is the AU site corresponding to nucleotides 271-272.

23. A DNAzyme as claimed in claim 21 wherein the 3′-end nucleotide residue is inverted in the binding domain contiguous with the 3′-end of the catalytic domain.

24. A DNAzyme as claimed in claim 22 wherein the 3′-end nucleotide residue is inverted in the binding domain contiguous with the 3′-end of the catalytic domain.

25. A DNAzyme as claimed in claim 1 which has a sequence selected from the group consisting of:

(i) 5′-caggggacaGGCTAGCTACAACGAcgttgcggg (SEQ ID NO: 3);

(ii) 5′-tgcaggggaGGCTAGCTACAACGAaccgttgcg (SEQ ID NO: 4);

(iii) 5′-catcctggaGGCTAGCTACAACGAgagcaggct (SEQ ID NO: 5);

(iv) 5′-ccgcggccaGGCTAGCTACAACGAcctggacga (SEQ ID NO: 6);

(v) 5′-ccgctgccaGGCTAGCTACAACGAcccggacgt (SEQ ID NO: 7);

(vi) 5′-gcggggacaGGCTAGCTACAACGAcagctgcat (SEQ ID NO: 8);

(vii) 5′-cagcggggaGGCTAGCTACAACGAatcagctgc (SEQ ID NO: 9); and

(viii) 5′-ggtcagagaGGCTAGCTACAACGActgcagcgg (SEQ ID NO: 10).

26. A DNAzyme as claimed in claim 25 wherein the 3′-end nucleotide residue is inverted in the binding domain contiguous with the 3′-end of the catalytic domain.

27. A DNAzyme as claimed in claim 25 which has the sequence:

5′-ccgcggccaGGCTAGCTACAACCAcctggacga (SEQ ID NO: 6).

28. A DNAzyme as claimed in claim 27 wherein the 3′-end nucleotide residue is inverted in the binding domain contiguous with the 3′-end of the catalytic domain.

29. A pharmaceutical composition comprising a DNAzyme according to claim 1 and a pharmaceutically acceptable carrier.