Human DNase I variants

• US 6,348,343 B2
• Filed: 02/28/2001
• Issued: 02/19/2002
• Est. Priority Date: 02/24/1995
• Status: Expired due to Term

• Current Assignee: Genentech, Inc. (Roche Holding AG)
• Original Assignee: Genentech, Inc. (Roche Holding AG)
• Inventors: Lazarus, Robert A., Ulmer, Jana S., Shak, Steven
• Primary Examiner(s): Patterson, Jr., Charles L.
• Application Number:
  US09/796,774
• Publication Number:
  US 20010041360A1
• Time in Patent Office: 356 Days
• Field of Search: 435/194, 424/94.6, 536/23.2
• US Class Current: 435/199
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

The present invention relates to amino acid sequence variants of human DNase I that have reduced binding affinity for actin. The invention provides nucleic acid sequences encoding such actin-resistant variants, thereby enabling the production of these variants in quantities sufficient for clinical use. The invention also relates to pharmaceutical compositions and therapeutic uses of actin-resistant variants of human DNase I.

Claims

1. A variant of human DNase I (SEQ ID NO:

1) comprising at least one amino acid substitution at the following positions corresponding to the sequence of native human DNase I;

His44, Leu45, Val48, Gly49, Leu52, Asp53, Asn56, His64, Tyr65, Val66, Val67, Ser68, Glu69, Ser94, Tyr96 or Ala 114, wherein said variant has DNA hydrolytic activity.

2. A variant of claim 1 that has a binding affinity for actin that is at least five-fold less than that of native human DNase I.

3. A variant of claim 1 that has a binding affinity for actin that is at least 100-fold less than that of native human DNase I.

5. A variant of claim 1 comprising an amino acid sequence having at least 95% identity with the amino acid sequence of native human DNase I (SEQ ID NO:

1).

6. A variant of claim 1 having an amino acid sequence that differs from the amino acid sequence of native human DNase I (SEQ ID NO:

1) by the substitution of one amino acid for another at only a single position within the sequence.

7. An isolated nucleic acid encoding a human DNase I variant according to claim 1.

8. The nucleic acid of claim 7 comprising a nucleotide sequence that encodes an amino acid sequence having at least 90% identity with the amino acid sequence of native human DNase I (SEQ ID NO:

1).

9. The nucleic acid of claim 7 comprising a nucleotide sequence that encodes an amino acid sequence having at least 95% identity with the amino acid sequence of native human DNase I (SEQ ID NO:

1).

10. The nucleic acid of claim 7 comprising a nucleotide sequence that encodes an amino acid sequence that differs from the amino acid sequence of native human DNase I (SEQ ID NO:

1) by the substitution of one amino acid for another at only a single position within the sequence.

11. A method of the treatment of a patient having a pulmonary disease or disorder comprising administering to the patient a therapeutically effective amount of a variant of human DNase I according to claim 1.

12. The method of claim 11 wherein the disease or disorder is cystic fibrosis.

13. The method of claim 11 wherein the disease or disorder is chronic bronchitis.

14. A pharmaceutical composition comprising a variant of human DNase I according to claim 1 and a pharmaceutically acceptable excipient.

15. The composition of claim 14 wherein the composition is in liquid form.

16. The composition of claim 15 wherein the composition is in powder form.

4. A variant of claim I comprising an amino acid sequence having at least 90% identity with the amino acid sequence of native human DNase I (SEQ ID NO:

1).

17. A variant of a human DNase I (SEQ ID NO:

1) comprising at least one amino acid substitution selected from the group consisting of E13A, E13H, E13R, E13W, E13Y, H44A, H44D, H44Y, H44W, H44C, H44Q, H44N, H44E, L45C, L45K, L45R, V48C, V48K, V48R, G49C, G491, G49K, G49R, G49Y, L52C, L52K, L52M, L52N, L52R, D53A, D53K, D53R, D53Y, D53C, D53L, D53M, N56C, N56F, N56K, N56R, N56W, D58T, H64N, Y65A, Y65R, Y65W, Y65C, Y65K, Y65M, Y65S, Y65N, Y65E, Y65P, V66T, V66N, V67A, V67E, V67K, V67C, V67D, V67H, V67M, V67P, V67R, V67S, V67T, V67N, S68K, S68R, S68M, S68N, E69K, E69R, E69A, E69C, E69M, E69T, P70T, S94N, Y96T, A114C, A114E, A114G, A114H, A114K, A114L, A114M, A114Q, A114R, A114W and A114Y, wherein said variant has DNA hydrolytic activity.

18. A variant according to claim 17, comprising at least one amino acid substitution selected from the group consisting of:

E13A, E13H, E13R, E13W, E13Y, H44A, G49R, D53R, D53K, D53Y, D53A, D53C, N56R, Y65A, Y65R, Y65W, V67E, E69K, E69R A114G and A114H.

19. A variant according to claim 17, comprising at least one amino acid substitution selected from the group consisting of:

H44A;

D53R;

Y65A, H44A;

Y65A;

E69R, D53R;

Y65A, D53R;

E69R, S94N;

Y96T, V67N;

E69T, Y65N;

V67T and H64N;

V66T.

20. A variant of claim 17 comprising an amino acid sequence having at least 90% identity with the amino acid sequence of native human DNase I (SEQ ID NO:

1).

21. A variant of claim 17 comprising an amino acid sequence having at least 95% identity with the amino acid sequence of native human DNase I (SEQ ID NO:

1).