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

• US 20070117128A1
• Filed: 10/18/2006
• Published: 05/24/2007
• Est. Priority Date: 10/18/2005
• Status: Active Grant

• Current Assignee: Duke University
• Original Assignee: Duke University
• Inventors: Jantz, Derek, Smith, James, Hellinga, Homme
• Granted Patent: US 8,021,867 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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

1-48. -48. (canceled)

49. A recombinant meganuclease having altered specificity for at least one recognition sequence half-site relative to a wild-type I-CreI meganuclease, comprising:

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

1; and

having specificity for a recognition sequence half-site which differs by at least one base pair from a half-site within an I-CreI meganuclease recognition sequence selected from the group consisting of SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4 and SEQ ID NO;

5;

wherein;

(l) specificity at position −

1 has been altered;

(a) to a T on a sense strand by a modification comprising Q70;

or

(b) to any base on a sense strand by a modification selected from the group consisting of A70 and S70; and

/or

(2) specificity at position −

2 has been altered;

(a) to an A on a sense strand by a modification selected from the group consisting of Q70, T44 and N44;

or

(b) to a C on a sense strand by a modification comprising D70;

or

(c) to an A or T on a sense strand by a modification comprising C44; and

/or

(3) specificity at position −

3 has been altered;

(a) to an A on a sense strand by a modification comprising C24;

or

(b) to a C on a sense strand by a modification selected from the group consisting of E68 and K24;

or

(c) to an A or C on a sense strand by a modification comprising H68;

or

(d) to a C or T on a sense strand by a modification comprising Y68;

or

(e) to a G or T on a sense strand by a modification comprising K68; and

/or

(4) specificity at position −

4 has been altered;

(a) to a C on a sense strand by a modification comprising E77;

or

(b) to a G on a sense strand by a modification selected from the group consisting of E26 and R77;

or

(c) to an A on a sense strand by a modification comprising Q77;

or

(d) to a C or T on a sense strand by a modification comprising S77;

or

(e) to any base on a sense strand by a modification comprising S26; and

/or

(5) specificity at position −

5 has been altered;

(a) to a C on a sense strand by a modification comprising E42;

or

(b) to a G on a sense strand by a modification comprising R42;

or

(c) to an A or G on a sense strand by a modification selected from the group consisting of C28 and Q42;

or

(d) to any base on a sense strand by a modification selected from the group consisting of M66 and K66; and

/or

(6) specificity at position −

6 has been altered;

(a) to an A on a sense strand by a modification selected from the group consisting of Q40 and C28;

or

(b) to a C on a sense strand by a modification selected from the group consisting of E40 and R28;

or

(c) to a G on a sense strand by a modification comprising R40; and

/or

(7) specificity at position −

7 has been altered;

(a) to a C on a sense strand by a modification selected from the group consisting of E38 and R30;

or

(b) to a G on a sense strand by a modification selected from the group consisting of R38 and E30; and

/or

(8) specificity at position −

8 has been altered;

(a) to a G on a sense strand by a modification comprising K33; and

/or

(9) specificity at position −

9 has been altered;

(a) to a T on a sense strand by a modification comprising C32;

or

(b) to a C or T on a sense strand by a modification comprising D32;

or

(c) to any base on a sense strand by a modification comprising N32.

50. The recombinant meganuclease of claim 49, further comprising:

at least one modification whereby DNA-binding affinity has been increased corresponding to a substitution selected from the group consisting of;

(a) substitution of E80, D137, I81, L112, P29, V64 or Y66 with H, N, Q, S, T, K or R;

or

(b) substitution of T46, T140 or T143 with K or R;

or

at least one modification whereby DNA-binding affinity has been decreased corresponding to a substitution selected from the group consisting of;

(a) substitution of K34, K48, R51, K82, K116 or K139 with H, N, Q, S, T, D or E;

or

(b) substitution of I81, L112, P29, V64, Y66, T46, T140 or T143 with D or E.

51. The recombinant meganuclease of claim 49, further comprising:

at least one modification whereby affinity for dimer formation has been altered corresponding to a substitution selected from the group consisting of;

(a) substitution of K7, K57 or K96 with D or E;

or

(b) substitution of E8 or E61 with K or R.

52. A recombinant meganuclease having altered binding affinity for double-stranded DNA relative to a wild-type I-CreI meganuclease, comprising:

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

1;

wherein DNA-binding affinity has been increased by at least one modification corresponding to a substitution selected from the group consisting of;

(a) substitution of E80, D137, I81, L112, P29, V64 or Y66 with H, N, Q, S, T, K or R;

or

(b) substitution of T46, T140 or T143 with K or R;

or

wherein DNA-binding affinity has been decreased by at least one modification corresponding to a substitution selected from the group consisting of;

(a) substitution of K34, K48, R51, K82, K116 or K139 with H, N, Q, S, T, D or E;

or

(b) substitution of I81, L112, P29, V64, Y66, T46, T140 or T143 with D or E.

53. A recombinant meganuclease of claim 52, further comprising:

at least one modification selected from Table 1.

54. The recombinant meganuclease of claim 52, further comprising:

at least one modification whereby affinity for dimer formation has been altered corresponding to a substitution selected from the group consisting of;

(a) substitution of K7, K57 or K96 with D or E;

or

(b) substitution of E8 or E61 with K or R.

55. A recombinant meganuclease monomer having altered affinity for dimer formation with a reference meganuclease monomer, comprising:

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

1;

wherein affinity for dimer formation has been altered by at least one modification corresponding to a substitution selected from the group consisting of;

(a) substitution of K7, K57 or K96 with D or E;

or

(b) substitution of E8 or E61 with K or R.

56. A recombinant meganuclease of claim 55, further comprising:

at least one modification selected from Table 1.

57. A recombinant meganuclease of claim 55, further comprising:

at least one modification whereby DNA-binding affinity has been increased corresponding to a substitution selected from the group consisting of;

(a) substitution of E80, D137, I81, L112, P29, V64 or Y66 with H, N, Q, S, T, K or R;

or

(b) substitution of T46, T140 or T143 with K or R;

or

at least one modification whereby DNA-binding affinity has been decreased corresponding to a substitution selected from the group consisting of;

(a) substitution of K34, K48, R51, K82, K116 or K139 with H, N, Q, S, T, D or E;

or

(b) substitution of I81, L112, P29, V64, Y66, T46, T140 or T143 with D or E.

58. A recombinant meganuclease heterodimer comprising:

a first polypeptide having at least 85% sequence similarity to residues 2-153 of the I-CreI meganuclease of SEQ ID NO;

1;

wherein affinity for dimer formation has been altered by at least one modification corresponding to a substitution selected from the group consisting of;

(a) substitution of K7, K57 or K96 with D or E; and

a second polypeptide having at least 85% sequence similarity to residues 2-153 of the I-CreI meganuclease of SEQ ID NO;

1;

wherein affinity for dimer formation has been altered by at least one modification corresponding to a substitution selected from the group consisting of;

(b) substitution of E8 or E61 with K or R.

59. A recombinant meganuclease of claim 58, further comprising:

at least one modification selected from Table 1.

60. A recombinant meganuclease of claim 58, further comprising:

at least one modification whereby DNA-binding affinity has been increased corresponding to a substitution selected from the group consisting of;

(a) substitution of E80, D137, I81, L112, P29, V64 or Y66 with H, N, Q, S, T, K or R;

or

(b) substitution of T46, T140 or T143 with K or R;

or

at least one modification whereby DNA-binding affinity has been decreased corresponding to a substitution selected from the group consisting of;

(a) substitution of K34, K48, R51, K82, K116 or K139 with H, N, Q, S, T, D or E;

or

(b) substitution of I81, L112, P29, V64, Y66, T46, T140 or T143 with D or E.