RATIONALLY-DESIGNED MEGANUCLEASES WITH ALTERED SEQUENCE SPECIFICITY AND DNA-BINDING AFFINITY

US 20120021465A1
Filed: 09/27/2011
Published: 01/26/2012
Est. Priority Date: 10/18/2005
Status: Active Grant

First Claim

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1. A method of cleaving a target recognition sequence in double-stranded DNA, wherein the target sequence differs by at least one nucleotide modification from the wild-type I-CreI meganuclease recognition sequence of SEQ ID NO:

4, including a substitution corresponding to position −

9 of the I-CreI recognition site, the method comprising;

(a) providing a recombinant meganuclease, or an expression vector encoding the recombinant meganuclease, wherein;

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

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

1 by 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

wherein;

(i) if the nucleotide corresponding to position −

9 of the I-CreI meganuclease recognition sequence has been altered to a G on a sense strand, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;

1 selected from the group consisting of S32R, S32K, S32N, S32H, S32Q and S32T;

(ii) if the nucleotide corresponding to position −

9 of the I-CreI meganuclease recognition sequence has been altered to an T on a sense strand, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;

1 selected from the group consisting of S32L, S32V, S32A, S32C, S32D, S32I, S32N, S32H, S32Q and S32T;

(iii) if the nucleotide corresponding to position −

9 of the I-CreI meganuclease recognition sequence has been altered to a A on a sense strand, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;

1 selected from the group consisting of S32N, S32H, S32Q and S32T; and

(b) contacting the recombinant meganuclease with the double-stranded DNA, whereby the meganuclease cleaves the target recognition sequence.

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Abstract

Rationally-designed LAGLIDADG 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.

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24 Claims

1. A method of cleaving a target recognition sequence in double-stranded DNA, wherein the target sequence differs by at least one nucleotide modification from the wild-type I-CreI meganuclease recognition sequence of SEQ ID NO:
- 4, including a substitution corresponding to position −
  
  9 of the I-CreI recognition site, the method comprising;
  
  (a) providing a recombinant meganuclease, or an expression vector encoding the recombinant meganuclease, wherein;
  
  the recombinant meganuclease comprises a polypeptide having at least 85% sequence identity to residues 2-153 of the I-CreI meganuclease of SEQ ID NO;
  
  1, and which differs from residues 2-153 of the I-CreI meganuclease of SEQ ID NO;
  
  1 by 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
  
  wherein;
  
  (i) if the nucleotide corresponding to position −
  
  9 of the I-CreI meganuclease recognition sequence has been altered to a G on a sense strand, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
  
  1 selected from the group consisting of S32R, S32K, S32N, S32H, S32Q and S32T;
  
  (ii) if the nucleotide corresponding to position −
  
  9 of the I-CreI meganuclease recognition sequence has been altered to an T on a sense strand, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
  
  1 selected from the group consisting of S32L, S32V, S32A, S32C, S32D, S32I, S32N, S32H, S32Q and S32T;
  
  (iii) if the nucleotide corresponding to position −
  
  9 of the I-CreI meganuclease recognition sequence has been altered to a A on a sense strand, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
  
  1 selected from the group consisting of S32N, S32H, S32Q and S32T; and
  
  (b) contacting the recombinant meganuclease with the double-stranded DNA, whereby the meganuclease cleaves the target recognition sequence.
- View Dependent Claims (23)
- - 23. The method of claim 1, wherein the double-stranded DNA is present in a cell, and the step of contacting comprises introducing the meganuclease into the cell or introducing a vector that expresses the meganuclease into the cell.

2. A method of cleaving a target recognition sequence in double-stranded DNA, wherein the target sequence differs by at least one nucleotide modification in the wild-type I-CreI meganuclease recognition sequence of SEQ ID NO:
- 4, including a substitution corresponding to position −
  
  9 of the I-CreI recognition site, the method comprising;
  
  (a) providing a recombinant meganuclease, or an expression vector encoding the recombinant meganuclease,wherein;
  
  the recombinant meganuclease comprises a polypeptide having at least 85% sequence identity to residues 2-153 of the I-CreI meganuclease of SEQ ID NO;
  
  1, and which differs from residues 2-153 of the I-CreI meganuclease of SEQ ID NO;
  
  1 by 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;
  
  wherein;
  
  (i) if the nucleotide corresponding to position −
  
  9 of the I-CreI meganuclease recognition sequence has been altered to a G on a sense strand, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
  
  1 selected from the group consisting of S32R, S32K, S32N, S32H, S32Q and S32T;
  
  (ii) if the nucleotide corresponding to position −
  
  9 of the I-CreI meganuclease recognition sequence has been altered to an T on a sense strand, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
  
  1 selected from the group consisting of S32L, S32V, S32A, S32C, S32D, S32I, S32N, S32H, S32Q and S32T;
  
  (iii) if the nucleotide corresponding to position −
  
  9 of the I-CreI meganuclease recognition sequence has been altered to a A on a sense strand, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
  
  1 selected from the group consisting of S32N, S32H, S32Q and S32T; and
  
  wherein;
  
  the recombinant meganuclease comprises at least a second specificity-altering amino acid modification corresponding to a substitution in SEQ ID NO;
  
  1 selected from the group consisting of;
  
  I24C, I24K, I24R, Q26A, Q26E, Q26K, Q26S, K28A, K28C, K28H, K28Q, K28R, K28S, N30E, N30K, N30Q, N30R, S32A, S32C, S32D, S32E, S32H, S32I, S32K, S32L, S32N, S32Q, S32R, S32T, S32V, Y33C, Y33D, Y33E, Y33F, Y33H, Y33I, Y33L, Y33R, Y33V, Q38C, Q38E, Q38H, Q38I, Q38K, Q38L, Q38N, Q38R, S40A, S40C, S40E, S40I, S40Q, 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, I77E, I77Q, I77R, I77S, S79A, S79C, S79I, S79V, K139H and K139Y; and
  
  (b) contacting the recombinant meganuclease with the double-stranded DNA, whereby the meganuclease cleaves the target recognition sequence.
- View Dependent Claims (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 24)
- - 3. The methods of claim 2 wherein the nucleotide corresponding to position −
    - 9 of the I-CreI meganuclease recognition site has been altered to a G on a sense strand and the first specificity-altering amino acid modification corresponds to a substitution of S32R in SEQ ID NO;
      
      1.
  - 4. The methods of claim 2 wherein the nucleotide corresponding to position −
    - 9 of the I-CreI meganuclease recognition site has been altered to a G on a sense strand and the first specificity-altering amino acid modification corresponds to a substitution of S32K in SEQ ID NO;
      
      1.
  - 5. The methods of claim 2 wherein the nucleotide corresponding to position −
    - 9 of the I-CreI meganuclease recognition site has been altered to a G on a sense strand and the first specificity-altering amino acid modification corresponds to a substitution of S32N in SEQ ID NO;
      
      1.
  - 6. The methods of claim 2 wherein the nucleotide corresponding to position −
    - 9 of the I-CreI meganuclease recognition site has been altered to a G on a sense strand and the first specificity-altering amino acid modification corresponds to a substitution of S32H in SEQ ID NO;
      
      1.
  - 7. The methods of claim 2 wherein the nucleotide corresponding to position −
    - 9 of the I-CreI meganuclease recognition site has been altered to a G on a sense strand and the first specificity-altering amino acid modification corresponds to a substitution of S32Q in SEQ ID NO;
      
      1.
  - 8. The methods of claim 2 wherein the nucleotide corresponding to position −
    - 9 of the I-CreI meganuclease recognition site has been altered to a G on a sense strand and the first specificity-altering amino acid modification corresponds to a substitution of S32T in SEQ ID NO;
      
      1.
  - 9. The methods of claim 2 wherein the nucleotide corresponding to position −
    - 9 of the I-CreI meganuclease recognition site has been altered to a T on a sense strand and the first specificity-altering amino acid modification corresponds to a substitution of S32L in SEQ ID NO;
      
      1.
  - 10. The methods of claim 2 wherein the nucleotide corresponding to position −
    - 9 of the I-CreI meganuclease recognition site has been altered to a T on a sense strand and the first specificity-altering amino acid modification corresponds to a substitution of S32V in SEQ ID NO;
      
      1.
  - 11. The methods of claim 2 wherein the nucleotide corresponding to position −
    - 9 of the I-CreI meganuclease recognition site has been altered to a T on a sense strand and the first specificity-altering amino acid modification corresponds to a substitution of S32A in SEQ ID NO;
      
      1.
  - 12. The methods of claim 2 wherein the nucleotide corresponding to position −
    - 9 of the I-CreI meganuclease recognition site has been altered to a T on a sense strand and the first specificity-altering amino acid modification corresponds to a substitution of S32C in SEQ ID NO;
      
      1.
  - 13. The methods of claim 2 wherein the nucleotide corresponding to position −
    - 9 of the I-CreI meganuclease recognition site has been altered to a T on a sense strand and the first specificity-altering amino acid modification corresponds to a substitution of S32D in SEQ ID NO;
      
      1.
  - 14. The methods of claim 2 wherein the nucleotide corresponding to position −
    - 9 of the I-CreI meganuclease recognition site has been altered to a T on a sense strand and the first specificity-altering amino acid modification corresponds to a substitution of S32I in SEQ ID NO;
      
      1.
  - 15. The methods of claim 2 wherein the nucleotide corresponding to position −
    - 9 of the I-CreI meganuclease recognition site has been altered to a T on a sense strand and the first specificity-altering amino acid modification corresponds to a substitution of S32N in SEQ ID NO;
      
      1.
  - 16. The methods of claim 2 wherein the nucleotide corresponding to position −
    - 9 of the I-CreI meganuclease recognition site has been altered to a T on a sense strand and the first specificity-altering amino acid modification corresponds to a substitution of S32H in SEQ ID NO;
      
      1.
  - 17. The methods of claim 2 wherein the nucleotide corresponding to position −
    - 9 of the I-CreI meganuclease recognition site has been altered to a T on a sense strand and the first specificity-altering amino acid modification corresponds to a substitution of S32Q in SEQ ID NO;
      
      1.
  - 18. The methods of claim 2 wherein the nucleotide corresponding to position −
    - 9 of the I-CreI meganuclease recognition site has been altered to a T on a sense strand and the first specificity-altering amino acid modification corresponds to a substitution of S32T in SEQ ID NO;
      
      1.
  - 19. The methods of claim 2 wherein the nucleotide corresponding to position −
    - 9 of the I-CreI meganuclease recognition site has been altered to an A on a sense strand and the first specificity-altering amino acid modification corresponds to a substitution of S32N in SEQ ID NO;
      
      1.
  - 20. The methods of claim 2 wherein the nucleotide corresponding to position −
    - 9 of the I-CreI meganuclease recognition site has been altered to an A on a sense strand and the first specificity-altering amino acid modification corresponds to a substitution of S32H in SEQ ID NO;
      
      1.
  - 21. The methods of claim 2 wherein the nucleotide corresponding to position −
    - 9 of the I-CreI meganuclease recognition site has been altered to an A on a sense strand and the first specificity-altering amino acid modification corresponds to a substitution of S32Q in SEQ ID NO;
      
      1.
  - 22. The methods of claim 2 wherein the nucleotide corresponding to position −
    - 9 of the I-CreI meganuclease recognition site has been altered to an A on a sense strand and the first specificity-altering amino acid modification corresponds to a substitution of S32T in SEQ ID NO;
      
      1.
  - 24. The method of claim 2, wherein the double-stranded DNA is present in a cell, and the step of contacting comprises introducing the meganuclease into the cell or introducing a vector that expresses the meganuclease into the cell.

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Current Assignee
Duke University
Original Assignee
Duke University
Inventors
Smith, James J., Jantz, Derek, Hellinga, Homme W.

Granted Patent

US 8,124,369 B2
Time in Patent Office

Days
Field of Search
US Class Current

435/91.53
CPC Class Codes

A61K 38/465   acting on ester bonds (3.1)...

A61K 48/00   Medicinal preparations cont...

A61P 3/00   Drugs for disorders of the ...

A61P 31/00   Antiinfectives, i.e. antibi...

A61P 31/12   Antivirals

A61P 43/00   Drugs for specific purposes...

C12N 15/8213   Targeted insertion of genes...

C12N 15/902   using homologous recombination

C12N 15/905   in yeast

C12N 15/907   in mammalian cells

C12N 9/22   Ribonucleases RNAses, DNAse...

RATIONALLY-DESIGNED MEGANUCLEASES WITH ALTERED SEQUENCE SPECIFICITY AND DNA-BINDING AFFINITY

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RATIONALLY-DESIGNED MEGANUCLEASES WITH ALTERED SEQUENCE SPECIFICITY AND DNA-BINDING AFFINITY

First Claim

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Abstract

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24 Claims

Specification

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