Rationally-designed meganucleases with altered sequence specificity and DNA-binding affinity

• US 8,021,867 B2
• Filed: 10/18/2006
• Issued: 09/20/2011
• Est. Priority Date: 10/18/2005
• Status: Active Grant

• Current Assignee: Duke University
• Original Assignee: Duke University
• Inventors: Hellinga, Homme W., Smith, James J., Jantz, Derek
• Primary Examiner(s): RAMIREZ, DELIA M
• Application Number:
  US11/583,368
• Publication Number:
  US 20070117128A1
• Time in Patent Office: 1,798 Days
• Field of Search: 435/196, 435/69.1, 435/325, 435/320.1, 435/252.3, 435/6, 530/350, 536/23.2
• US Class Current: 435/196
• CPC Class Codes: A61K 38/465 acting on ester bonds (3.1), e.g. lipases, ribonucleases
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 15/8213 Targeted insertion of genes into the plant genome by homologous recombination
• CPC Class Codes: C12N 15/902 using homologous recombination
• CPC Class Codes: C12N 15/905 in yeast
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

Rationally-designed LAGLIDADG (SEQ ID NO: 37) meganucleases and methods of making such meganucleases are provided. In addition, methods are provided for using the meganucleases to generate recombinant cells and organisms having a desired DNA sequence inserted into a limited number of loci within the genome, as well as methods of gene therapy, for treatment of pathogenic infections, and for in vitro applications in diagnostics and research.

Claims

1. A recombinant meganuclease having altered cleavage specificity relative to the wild-type I-CreI meganuclease of SEQ ID NO:

1, comprising a polypeptide having at least 85% sequence identity to residues 2-153 of the I-CreI meganuclease of SEQ ID NO;

1, which differs from residues 2-153 of the I-CreI meganuclease of SEQ ID NO;

1 by;

(a) at least a first specificity-altering amino acid modification at a position corresponding to a position of SEQ ID NO;

1 selected from the group consisting of positions 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 66, 68, 70, 75, 77, 79 and 139; and

(b) at least a second specificity-altering amino acid modification at a position corresponding to position 26 or 77 of SEQ ID NO;

1, wherein said second specificity-altering amino acid modification is a substitution selected from the group consisting of Q26E, Q26K, Q26S, I77E, I77Q, I77R and I77S; and

(c) at least a third amino acid modification affecting heterodimer formation selected from the group consisting of K7D, K7E, E8K, E8R, K57D, K57E, E61K, E61R, K96D and K96E.

3. The recombinant meganuclease of claim 1 or 2 wherein:

said second amino acid modification is Q26E.

4. The recombinant meganuclease of claim 1 or 2 wherein:

said second amino acid modification is Q26K.

5. The recombinant meganuclease of claim 1 or 2 wherein:

said second amino acid modification is Q26S.

6. The recombinant meganuclease of claim 1 or 2 wherein:

said second amino acid modification is I77E.

7. The recombinant meganuclease of claim 1 or 2 wherein:

said second amino acid modification is I77Q.

8. The recombinant meganuclease of claim 1 or 2 wherein:

said second amino acid modification is I77R.

9. The recombinant meganuclease of claim 1 or 2 wherein:

said second amino acid modification is I77S.

10. The recombinant meganuclease of claim 1 or 2 wherein:

said first amino acid modification is selected from the group consisting of I24C, I24K, I24R, K28C, K28Q, K28R, N30E, N30K, N30Q, N30R, S32A, S32C, S32D, S32E, S32H, S32I, S32K, S32L, S32N, S32Q, S32R, S32T, S32V, Y33C, Y33D, Y33E, Y33F, Y33I, Y33K, Y33L, Y33R, Y33V, Q38C, Q38E, Q38H, Q38I, Q38K, Q38L, Q38N, Q38R, S40C, S40E, S40I, S40R, S40V, A42E, A42Q, A42R, Q44A, Q44C, Q44D, Q44E, Q44I, Q44K, Q44L, Q44N, Q44R, Q44T, Q44V, T46A, T46C, T46D, T46E, T46G, T46H, T46K, T46Q, T46R, Y66K, Y66M, R68C, R68E, R68F, R68H, R68K, R68L, R68M, R68Q, R68Y, R70A, R70C, R70D, R70E, R70G, R70H, R70K, R70L, R70Q, R70S, D75C, D75E, D75H, D75L, D75Q, D75R, D75Y, S79C, S79I, S79V, K139H, and K139Y.

2. A recombinant meganuclease having altered cleavage specificity relative to the wild-type I-CreI meganuclease of SEQ ID NO:

1, comprising a polypeptide having at least 85% sequence identity to residues 2-153 of the I-CreI meganuclease of SEQ ID NO;

1, which differs from residues 2-153 of the I-CreI meganuclease of SEQ ID NO;

1 by;

(a) at least a first specificity-altering amino acid modification at a position corresponding to a position of SEQ ID NO;

1 selected from the group consisting of positions 24, 26, 28, 30, 32, 33, 38, 40, 42, 44, 46, 66, 68, 70, 75, 77, 79 and 139; and

(b) at least a second specificity-altering amino acid modification at a position corresponding to position 26 or 77 of SEQ ID NO;

1, wherein said second specificity-altering amino acid modification is a substitution selected from the group consisting of Q26E, Q26K, Q26S, I77E, I77Q, I77R and I77S; and

(c) at least a third amino acid modification affecting DNA binding affinity selected from the group consisting of P29D, P29E, P29H, P29K, P29N, P29Q, P29R, P29S, P29T, K34D, K34E, K34H, K34N, K34Q, K34S, K34T, K48D, K48E, K48H, K48N, K48Q, K48S, K48T, R51D, R51E, R51H, R51N, R51Q, R51S, R51T, V64D, V64E, V64H, V64K, V64N, V64Q, V64R, V64S, V64T, E80H, E80K, E80N, E80Q, E80R, E80S, E80T, I81D, I81E, I81H, I81K, I81N, I81Q, I81R, I81S, I81T, K82D, K82E, K82H, K82N, K82Q, K82S, K82T, L112D, L112E, L112K, L112H, L112N, L112Q, L112R, L112S, L112T, K116D, Kl116E, K116H, K116N, K116Q, K116S, K116T, D137H, D137K, D137N, D137Q, D137R, D137S, D137T, K139D, K139E, K139H, K139N, K139Q, K139S, K139T, T143D, T143E, T143K and T143R.

11. The recombinant meganuclease of claim 2 wherein:

said third amino acid modification is E80R.

12. The recombinant meganuclease of claim 2 wherein:

said third amino acid modification is E80K.

13. The recombinant meganuclease of claim 2 wherein:

said third amino acid modification is E80Q.

14. The recombinant meganuclease of claim 2 wherein:

said third amino acid modification is E80H.

15. The recombinant meganuclease of claim 2 wherein:

said third amino acid modification is E80N.

16. The recombinant meganuclease of claim 2 wherein:

said third amino acid modification is E80S.

17. The recombinant meganuclease of claim 2 wherein:

said third amino acid modification is E80T.

18. The recombinant meganuclease of claim 2 wherein:

said third amino acid modification is I81K.

19. The recombinant meganuclease of claim 2 wherein:

said third amino acid modification is I81R.