Gene repair involving the induction of double-stranded DNA cleavage at a chromosomal target site
AssigneesChildren's Medical Center Corporation, Institute Pasteur
InventorsAndre Choulika, Richard C. Mulligan
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Abstract
Methods of modifying, repairing, attenuating and inactivating a gene or other chromosomal DNA in a cell are disclosed. Also disclosed are methods of treating or prophylaxis of a genetic disease in an individual in need thereof. Further disclosed are chimeric restriction endonucleases.
Claims52
A method of repairing a specific sequence of interest in chromosomal DNA of a cell comprising the steps of:
- (a) inducing in the cell double stranded cleavage at a site of interest using a chimeric restriction endonuclease, said chimeric restriction endonuclease comprising a DNA binding sequence and a DNA cleavage domain;
- and
- (b) introducing targeting DNA into the cell under conditions appropriate for introduction of the targeting DNA into the site of interest, wherein said targeting DNA comprises (1) DNA homologous to the region surrounding the site of interest and (2) DNA which repairs the specific sequence of interest upon recombination between said targeting DNA and the chromosomal DNA.
A method of modifying a specific sequence in chromosomal DNA of a cell comprising the steps of:
- (a) inducing in the cell double stranded cleavage at a site of interest in the specific sequence to be modified using a chimeric restriction endonuclease, said chimeric restriction endonuclease comprising a DNA binding sequence and a DNA cleavage domain;
- and
- (b) introducing targeting DNA into the cell under conditions appropriate for introduction of the targeting DNA into the site of interest, wherein said targeting DNA comprises (1) DNA homologous to the region surrounding the site of interest and (2) DNA which modifies the specific sequence upon recombination between said targeting DNA and the chromosomal DNA.
A method of attenuating an endogenous gene of interest in a cell comprising the steps of:
- (a) inducing in the cell double stranded cleavage at a site of interest in the endogenous gene of interest using a chimeric restriction endonuclease, said chimeric restriction endonuclease comprising a DNA binding sequence and a DNA cleavage domain;
- and
- (b) introducing targeting DNA into the cell under conditions appropriate for introduction of the targeting DNA into the site of interest, wherein said targeting DNA comprises (1) DNA homologous to the region surrounding the site of interest and (2) DNA which attenuates the gene of interest upon recombination between said target DNA and the gene of interest.
A method of introducing a mutation into a site of interest in of chromosomal DNA of a cell comprising the steps of:
- (a) inducing in the cell double stranded cleavage at the site of interest using a chimeric restriction endonuclease, said chimeric restriction endonuclease comprising a DNA binding sequence and a DNA cleavage domain;
- and
- (b) introducing targeting DNA into the cell under conditions appropriate for introduction of the targeting DNA into the site of interest, wherein said targeting DNA comprises (1) DNA homologous to the region surrounding the site of interest and (2) the mutation to be introduced into the site of interest.
A method for treating or prophylaxis of a genetic disease in an individual in need thereof comprising the steps of:
- (a) inducing in cells of the individual double stranded cleavage at a site of interest using a chimeric restriction endonuclease, said chimeric restriction endonuclease comprising a DNA binding sequence and a DNA cleavage domain;
- and
- (b) introducing targeting DNA into the individual under conditions appropriate for introduction of the targeting DNA into the site of interest, wherein said targeting DNA comprises (1) DNA homologous to the region surrounding the site of interest and (2) DNA which repairs the site of interest upon recombination between the targeting DNA and the chromosomal DNA.
A method for treating or prophylaxis of a genetic disease in an individual in need thereof comprising inducing in cells of the individual double stranded cleavage at a site of interest using a chimeric restriction endonuclease, said chimeric restriction endonuclease comprising a DNA binding sequence and a DNA cleavage domain, under conditions appropriate for chromosomal DNA homologous to the region surrounding the site of interest to be introduced into the site of interest and repair of the site of interest.
A method of correcting a genetic lesion in chromosomal DNA of a cell comprising inducing in the cell double stranded cleavage at a site of interest in the genetic lesion using a chimeric restriction endonuclease, said chimeric restriction endonuclease comprising a DNA binding sequence and a DNA cleavage domain, under conditions appropriate for chromosomal DNA homologous to the region surrounding the site of interest to be introduced into the site of interest and correction of the genetic lesion.
A method of modifying a specific sequence in chromosomal DNA of a cell comprising inducing in the cell double stranded cleavage at a site of interest in the specific sequence to be modified using a chimeric restriction endonuclease, said chimeric restriction endonuclease comprising a DNA binding sequence and a DNA cleavage domain, under conditions appropriate for chromosomal DNA homologous to the region surrounding the site of interest to be introduced into the site of interest and modification of the specific sequence.
A method of repairing a specific sequence of interest in chromosomal DNA of a cell comprising inducing in the cell double stranded cleavage at a site of interest using a chimeric restriction endonuclease, said chimeric restriction endonuclease comprising a DNA binding sequence and a DNA cleavage domain, under conditions appropriate for chromosomal DNA homologous to the region surrounding the site of interest to be introduced into the site of interest and repair of the specific sequence of interest.
A chimeric restriction endonuclease comprising a DNA binding sequence and a DNA cleavage domain.
Dependencies are inferred from the claim text.