Method for genome modification

Abstract

The present disclosure includes methods for manipulation of the genome, to products obtained or obtainable from such methods, and uses of these products.

1 . A method .of modifying the genome of a cell, the method comprising recombining into the genome of the cell a nucleic acid, the exogenous nucleic acid comprising
 (i) a modifying sequence and   (ii) a selection element flanked by transposon inverted repeats,   the method further comprising the steps of:   (a) selection of insertion of the nucleic acid into the genome using the selection element, and   (b) use of a transposase to excise the selection element, such that after transposase mediated excision the only exogenous nucleic acid remaining in the genome is the modifying sequence   
     
     
         2 . A method of modifying according to  claim 1  wherein the modification is correcting or introducing a mutation in a genome of a cell, wherein the modifying sequence comprises nucleic acid which corrects or introduces the mutation, respectively, and wherein after the transposon mediated excision process the genome of the corrected cell is identical to the genome of the original cell with the only exceptions being the mono or biallelic correction of the mutation or mono or biallelic introduction of the mutation. 
     
     
         3 . A method according to  claim 1  or  2  comprising an additional step of providing to the cell a Zinc Finger Nucleases (ZFNs) or Transcription activator like effector nuclease (TALES) to increase efficiency of the homologous recombination of the nucleic acid into the cell genome. 
     
     
         4 . A method according to any preceding claim wherein the transposon sequences are from the piggyback transposon. 
     
     
         5 . A method according to any preceding  claim 1  wherein the transposase used to excise the nucleic acid comprising the selectable element is a piggyBac transposase. 
     
     
         6 . A method according to any preceding claim wherein the cell is an iPS cell from an individual having a genomic mutation associated with disease. 
     
     
         7 . A method according to any preceding claim wherein the modification is the correction of a point mutation. 
     
     
         8 . A method according to any preceding claim wherein modification is of one allele of a gene. 
     
     
         9 . A method according to any preceding claim wherein modification is simultaneous modification of both alleles of a gene. 
     
     
         10 . A method according to any preceding claim wherein the method provides at least 40% efficiency in targeting on one allele, determined as number of single allele modifications for cells having selected insertion of a selectable marker. 
     
     
         11 . A method according to any preceding claim wherein the method provides an efficiency of at least 3% biallelic targeting, determined as number of biallelic modifications for cells having selected insertion of a selectable marker. 
     
     
         12 . A method according to any preceding claim which is a method of modification of the genome of an iPS cell, the genome of which has a mutation associated with a disease phenotype, the method comprising delivering to the cell:
 (a) an exogenous nucleic acid comprising:   (i) a modifying sequence and   (ii) a selection element flanked by piggyBac inverted repeats; and   (b) a zinc finger nuclease or TALES to enhance the efficiency of homologous recombination of the nucleic acid into the genome of the cell;   wherein delivery is followed by selection for the insertion of the nucleic acid into the genome using the selection element; and then   providing expression of a piggyBac transposase within the cell to remove the selection element, optionally followed by differentiation of the modified cell.   
     
     
         13 . A cell comprising a genome which has been modified by insertion of a modifying sequence using a method according to  claim 1 - 12 . 
     
     
         14 . A collection of cells, such as an organ or tissue, wherein the genomes of all, or a proportion, of the cells has been modified by insertion of a modifying sequence using a method according to  claim 1 - 12 . 
     
     
         15 . A pair of cells, or cell lines or tissues differentiated therefrom, the genome of one of the pair having been modified from the other according to the method of any of  claims 1 - 12 . 
     
     
         16 . A cell, or cell line or tissues differentiated therefrom, made according to any of  claims 1 - 12 , optionally in combination with the original unmodified cell, for use in the assessment of any of the following:
 the effect of a therapeutic agent (e.g.drug screening);   the identification of targets for drug treatment;   the toxicological effect of a therapeutic agent;   the safety of a therapeutic agent;   the effect of an individual modification or modifications on drug activity;   the effect of an individual modification or modifications on likely patient responsiveness the effect of an individual modification or modifications on likely patient resistance;   in the assessment of which patient sub-groups will respond to drug treatments.   
     
     
         17 . A method for treating a disease in an individual, the method comprising modifying the genome of a cell using the method of  claims 1 - 12  to correct a mutation associated with the disease in the cell, and using the cell, or cells derived or differentiated therefrom, in disease treatment.