METHODS OF PRODUCING GENETICALLY-MODIFIED EUKARYOTIC CELLS WITH RATIONALLY-DESIGNED MEGANUCLEASES

US 20120266266A1
Filed: 06/25/2012
Published: 10/18/2012
Est. Priority Date: 10/18/2005
Status: Active Grant

First Claim

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1. A method for producing a genetically-modified eukaryotic non-human cell or an isolated human cell by modifying a target sequence in a chromosome of the cell, the method comprising:

introducing into the cell;

(i) a recombinant meganuclease, or (ii) a first nucleic acid that encodes a recombinant meganuclease and causes expression of the recombinant meganuclease in the cell;

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 the first specificity-altering amino acid modification is 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, Q381, Q38K, Q38L, Q38N, Q38R, S40A, S40C, S40E, S40I, S40Q, S40R, S40V, A42E, A42Q, A42R, Q44A, Q44C, Q44D, Q44E, Q441, 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;

wherein the DNA-binding affinity of the recombinant meganuclease has been altered compared to the DNA-binding affinity of the wild-type I-CreI meganuclease of SEQ ID NO;

1, and wherein the polypeptide further comprises at least one modification affecting DNA-binding affinity corresponding to a substitution in the polypeptide of SEQ ID NO;

1 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, L112H, L112K, L112N, L112Q, L112R, L112S, L112T, K116D, K116E, K116H, K116N, K116Q, K116S, K116T, D137H, D137K, D137N, D137Q, D137R, D137S, D137T, K139D, K139E, K139H, K139N, K139Q, K139S, K139T, T143D, T143E, T143K and T143R; and

wherein the recombinant meganuclease cleaves a site in the target sequence in the chromosome and the target sequence is modified by at least one base insertion, at least one base deletion, or at least one frameshift mutation in the chromosome.

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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.

1 Citation

27 Claims

1. A method for producing a genetically-modified eukaryotic non-human cell or an isolated human cell by modifying a target sequence in a chromosome of the cell, the method comprising:
- introducing into the cell;
  
  (i) a recombinant meganuclease, or (ii) a first nucleic acid that encodes a recombinant meganuclease and causes expression of the recombinant meganuclease in the cell;
  
  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 the first specificity-altering amino acid modification is 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, Q381, Q38K, Q38L, Q38N, Q38R, S40A, S40C, S40E, S40I, S40Q, S40R, S40V, A42E, A42Q, A42R, Q44A, Q44C, Q44D, Q44E, Q441, 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;
  
  wherein the DNA-binding affinity of the recombinant meganuclease has been altered compared to the DNA-binding affinity of the wild-type I-CreI meganuclease of SEQ ID NO;
  
  1, and wherein the polypeptide further comprises at least one modification affecting DNA-binding affinity corresponding to a substitution in the polypeptide of SEQ ID NO;
  
  1 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, L112H, L112K, L112N, L112Q, L112R, L112S, L112T, K116D, K116E, K116H, K116N, K116Q, K116S, K116T, D137H, D137K, D137N, D137Q, D137R, D137S, D137T, K139D, K139E, K139H, K139N, K139Q, K139S, K139T, T143D, T143E, T143K and T143R; and
  
  wherein the recombinant meganuclease cleaves a site in the target sequence in the chromosome and the target sequence is modified by at least one base insertion, at least one base deletion, or at least one frameshift mutation in the chromosome.
- View Dependent Claims (2, 3, 4, 5, 6)
- - 2. The method of claim 1, further comprising:
    - introducing into the cell a second nucleic acid comprising a sequence of interest;
      
      whereby the target sequence is modified by insertion of the sequence of interest into the chromosome.
  - 3. The method of claim 2, wherein:
    - the second nucleic acid further comprises sequences homologous to sequences flanking the site of cleavage and the sequence of interest is inserted into the chromosome by homologous recombination.
  - 4. The method of claim 2, wherein:
    - the second nucleic acid lacks substantial homology to sequences flanking the site of cleavage and the sequence of interest is inserted into the chromosome by non-homologous end-joining.
  - 5. The method of claim 1, further comprising growing the cell to produce a genetically-modified organism.
  - 6. The method of claim 5, wherein the cell is selected from the group consisting of a gamete, a zygote, a blastocyst cell, an embryonic stem cell, and a protoplast cell.

7. A method for producing a genetically-modified eukaryotic non-human cell or an isolated human cell by modifying a target sequence in a chromosome of the cell, the method comprising:
- introducing into the cell;
  
  (i) a recombinant meganuclease, or (ii) a first nucleic acid that encodes a recombinant meganuclease and causes expression of the recombinant meganuclease in the cell;
  
  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 the first specificity-altering amino acid modification is 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, Q381, Q38K, Q38L, Q38N, Q38R, S40A, S40C, S40E, S40I, S40Q, S40R, S40V, A42E, A42Q, A42R, Q44A, Q44C, Q44D, Q44E, Q441, 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;
  
  wherein the polypeptide further comprises a second amino acid modification affecting heterodimer formation selected from the group consisting of;
  
  K7D, K7E, E8K, E8R, K57D, K57E, E61K, E61R, K96D and K96E; and
  
  wherein the recombinant meganuclease cleaves a site in the target sequence in the chromosome and the target sequence is modified by at least one base insertion, at least one base deletion, or at least one frameshift mutation in the chromosome.
- View Dependent Claims (8, 9, 10, 11, 12)
- - 8. The method of claim 7, further comprising:
    - introducing into the cell a second nucleic acid comprising a sequence of interest;
      
      whereby the target sequence is modified by insertion of the sequence of interest into the chromosome.
  - 9. The method of claim 8, wherein:
    - the second nucleic acid further comprises sequences homologous to sequences flanking the site of cleavage and the sequence of interest is inserted into the chromosome by homologous recombination.
  - 10. The method of claim 8, wherein:
    - the second nucleic acid lacks substantial homology to sequences flanking the site of cleavage and the sequence of interest is inserted into the chromosome by non-homologous end-joining.
  - 11. The method of claim 7, further comprising growing the cell to produce a genetically-modified organism.
  - 12. The method of claim 11, wherein the cell is selected from the group consisting of a gamete, a zygote, a blastocyst cell, an embryonic stem cell, and a protoplast cell.

13. A method for producing a genetically-modified eukaryotic non-human cell or an isolated human cell by modifying a target sequence in a chromosome of the cell, the method comprising:
- introducing into the cell;
  
  (i) a recombinant meganuclease, or (ii) a first nucleic acid that encodes a recombinant meganuclease and causes expression of the recombinant meganuclease in the cell;
  
  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 the target sequence differs by at least one nucleotide modification from the wild-type I-CreI meganuclease recognition sequence of SEQ ID NO;
  
  4;
- View Dependent Claims (14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26)
- - 14. The method of claim 13, further comprising:
    - introducing into the cell a second nucleic acid comprising a sequence of interest;
      
      whereby the target sequence is modified by insertion of the sequence of interest into the chromosome.
  - 15. The method of claim 14, wherein:
    - the second nucleic acid further comprises sequences homologous to sequences flanking the site of cleavage and the sequence of interest is inserted at into the chromosome by homologous recombination.
  - 16. The method of claim 14, wherein:
    - the second nucleic acid lacks substantial homology to sequences flanking the site of cleavage and the sequence of interest is inserted into the chromosome by non-homologous end-joining.
  - 17. The method of claim 13, further comprising growing the cell to produce a genetically-modified organism.
  - 18. The method of claim 17, wherein the cell is selected from the group consisting of a gamete, a zygote, a blastocyst cell, an embryonic stem cell, and a protoplast cell.
  - 19. The method of claim 13, wherein the nucleotide modification corresponds to position −
    - 1 of the I-CreI recognition sequence of SEQ ID NO;
      
      4 and wherein;
      
      (i) if the nucleotide corresponding to position −
      
      1 of the I-CreI meganuclease recognition sequence of SEQ ID NO;
      
      4 has been altered to an A, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
      
      1 selected from the group consisting of D75C, D75L, D75Y, K139Y, T46A, T46C, T46G and R70A;
      
      (ii) if the nucleotide corresponding to position −
      
      1 of the I-CreI meganuclease recognition sequence of SEQ ID NO;
      
      4 has been altered to a G, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
      
      1 selected from the group consisting of T46E, T46D, R70E, D75E, T46G and R70A; and
      
      (iii) if the nucleotide corresponding to position −
      
      1 of the I-CreI meganuclease recognition sequence of SEQ ID NO;
      
      4 has been altered to a T, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
      
      1 selected from the group consisting of R70C, R70Q, D75H, D75Q, D75Y, K139H, T46H, T46Q, and T46G.
  - 20. The method of claim 13, wherein the nucleotide modification corresponds to position −
    - 3 of the I-CreI recognition sequence of SEQ ID NO;
      
      4 and wherein;
      
      (i) if the nucleotide corresponding to position −
      
      3 of the I-CreI meganuclease recognition sequence of SEQ ID NO;
      
      4 has been altered to an A, the specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
      
      1 of I24C; and
      
      (ii) if the nucleotide corresponding to position −
      
      3 of the I-CreI meganuclease recognition sequence of SEQ ID NO;
      
      4 has been altered to a C, the specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
      
      1 selected from the group consisting of I24K and I24R.
  - 21. The method of claim 13, wherein the nucleotide modification corresponds to position −
    - 4 of the I-CreI recognition sequence of SEQ ID NO;
      
      4 and wherein;
      
      (i) if the nucleotide corresponding to position −
      
      4 of the I-CreI meganuclease recognition sequence of SEQ ID NO;
      
      4 has been altered to a C, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
      
      1 selected from the group consisting of I77E, I77S, Q26K and Q26S;
      
      (ii) if the nucleotide corresponding to position −
      
      4 of the I-CreI meganuclease recognition sequence of SEQ ID NO;
      
      4 has been altered to a G, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
      
      1 selected from the group consisting of I77R, Q26E and Q26S; and
      
      (iii) if the nucleotide corresponding to position −
      
      4 of the I-CreI meganuclease recognition sequence of SEQ ID NO;
      
      4 has been altered to an A, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
      
      1 of Q26S.
  - 22. The method of claim 13, wherein the nucleotide modification corresponds to position −
    - 5 of the I-CreI recognition sequence of SEQ ID NO;
      
      4 and wherein;
      
      (i) if the nucleotide corresponding to position −
      
      5 of the I-CreI meganuclease recognition sequence of SEQ ID NO;
      
      4 has been altered to a G, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
      
      1 selected from the group consisting of A42Q, A42R, Y66M and Y66K;
      
      (ii) if the nucleotide corresponding to position −
      
      5 of the I-CreI meganuclease recognition sequence has been altered to an A, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
      
      1 selected from the group consisting of A42Q, Y66M, and Y66K; and
      
      (iii) if the nucleotide corresponding to position −
      
      5 of the I-CreI meganuclease recognition sequence of SEQ ID NO;
      
      4 has been altered to a T, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
      
      1 selected from the group consisting of Y66M and Y66K.
  - 23. The method of claim 13, wherein the nucleotide modification corresponds to position −
    - 6 of the I-CreI recognition sequence of SEQ ID NO;
      
      4 and wherein;
      
      (i) if the nucleotide corresponding to position −
      
      6 of the I-CreI meganuclease recognition sequence of SEQ ID NO;
      
      4 has been altered to a T, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
      
      1 selected from the group consisting of S40C, S40I, S40V, S79A, S79C, S79I, S79V and K28H;
      
      (ii) if the nucleotide corresponding to position −
      
      6 of the I-CreI meganuclease recognition sequence of SEQ ID NO;
      
      4 has been altered to a C, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
      
      1 of K28S; and
      
      (iii) if the nucleotide corresponding to position −
      
      6 of the I-CreI meganuclease recognition sequence of SEQ ID NO;
      
      4 has been altered to a G, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
      
      1 selected from the group consisting of S40R and K28S.
  - 24. The method of claim 13, wherein the nucleotide modification corresponds to position −
    - 7 of the I-CreI recognition sequence of SEQ ID NO;
      
      4 and wherein;
      
      (i) if the nucleotide corresponding to position −
      
      7 of the I-CreI meganuclease recognition sequence of SEQ ID NO;
      
      4 has been altered to a G, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
      
      1 of Q38C; and
      
      (ii) if the nucleotide corresponding to position −
      
      7 of the I-CreI meganuclease recognition sequence of SEQ ID NO;
      
      4 has been altered to a T, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
      
      1 selected from the group consisting of Q381 and Q38L.
  - 25. The method of claim 13, wherein the nucleotide modification corresponds to position −
    - 8 of the I-CreI recognition sequence of SEQ ID NO;
      
      4 and wherein;
      
      (i) if the nucleotide corresponding to position −
      
      8 of the I-CreI meganuclease recognition sequence of SEQ ID NO;
      
      4 has been altered to a C, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
      
      1 of S32R.
  - 26. The method of claim 13, wherein the nucleotide modification corresponds to position −
    - 9 of the I-CreI recognition sequence of SEQ ID NO;
      
      4 and wherein;
      
      (i) if the nucleotide corresponding to position −
      
      9 of the I-CreI meganuclease recognition sequence of SEQ ID NO;
      
      4 has been altered to a G, 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;
      
      (ii) if the nucleotide corresponding to position −
      
      9 of the I-CreI meganuclease recognition sequence of SEQ ID NO;
      
      4 has been altered to a T, the first specificity-altering amino acid modification corresponds to a substitution in SEQ ID NO;
      
      1 selected from the group consisting of S32L, S32V, S32C, S32D, S32I, S32N, S32H, S32Q and S32T; and
      
      (iii) if the nucleotide corresponding to position −
      
      9 of the I-CreI meganuclease recognition sequence of SEQ ID NO;
      
      4 has been altered to an A, 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.

27. A method for producing a genetically-modified eukaryotic non-human cell or an isolated human cell by modifying a target sequence in a chromosome of the cell, the method comprising:
- introducing into the cell;
  
  (i) a recombinant meganuclease, or (ii) a first nucleic acid that encodes a recombinant meganuclease and causes expression of the recombinant meganuclease in the cell;
  
  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 the target sequence differs by at least one nucleotide modification from the wild-type I-CreI meganuclease recognition sequence of SEQ ID NO;
  
  4;

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Current Assignee
Duke University
Original Assignee
Duke University
Inventors
SMITH, James J., JANTZ, Derek, HELLINGA, Homme W.

Granted Patent

US 8,377,674 B2
Time in Patent Office

Days
Field of Search
US Class Current

800/21
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...

METHODS OF PRODUCING GENETICALLY-MODIFIED EUKARYOTIC CELLS WITH RATIONALLY-DESIGNED MEGANUCLEASES

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METHODS OF PRODUCING GENETICALLY-MODIFIED EUKARYOTIC CELLS WITH RATIONALLY-DESIGNED MEGANUCLEASES

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