Tal effector means useful for partial or full deletion of dna tandem repeats

Abstract

The application relates to means, which derive from TAL effectors and TALENs. The structure of the means of the application is especially adapted for partial or full deletion of at least one DNA tandem repeat, more particularly for partial or full deletion of at least one DNA tandem repeat in a double-stranded DNA, more particularly for partial or full deletion of at least one DNA tandem repeat, which is contained in a double-stranded DNA and, which forms a complex secondary structure, such as a hairpin, a triple helix or a tetraplex secondary structure. The means of the application are notably useful in the treatment and/or prevention and/or palliation of a disease or disorder involving at least one DNA tandem repeat, such as DM1, SCA8, SCA12, HDL2, SBMA, HD, DRPLA, SCA1, SCA2, SCA3, SCA6, SCA7, SCA17, PSACH, DM2, SCA10, SPD1, OPMD, CCD, HPE5, HFG syndrome, BPES, EIEE1, FRAXA, FXTAS and FRAXE.

1 . A composition or kit comprising a first DNA-binding polypeptide and a second DNA-binding polypeptide,
 wherein said first DNA-binding polypeptide is different from said second DNA-binding polypeptide,   wherein each of said first and second DNA-binding polypeptides binds to a DNA nucleic acid comprising at least one DNA tandem repeat, wherein the DNA nucleic acid to which said first DNA-binding polypeptide binds is one strand of a double-stranded nucleic acid, wherein the DNA nucleic acid to which said second DNA-binding polypeptide binds is the other strand of the same double-stranded nucleic acid,   wherein said double-stranded DNA nucleic acid is a gene involved in a neurological and/or muscular and/or skeletal disorder or disease involving said at least one DNA tandem repeat,   wherein each of said first and second DNA-binding polypeptides comprises a TAL effector tandem repeat consisting of adjacent units of TAL effector tandem repeat,   wherein the ordered series of RVDs formed by the RVDs respectively contained in said adjacent units of TAL effector tandem repeat, in N- to C-orientation, is an ordered series of amino acids, which determines the recognition of the 5′-3′ nucleotide sequence of a DNA target site contained in the strand of double-stranded DNA nucleic acid to which said DNA-binding polypeptide binds,   wherein the sequence of said DNA target site is:
 i. a fragment of said strand of double-stranded DNA nucleic acid consisting of a fragment of said at least one DNA tandem repeat, wherein said fragment comprises more than one copy of said DNA sequence unit of said at least one DNA tandem repeat, or 
 ii. a fragment of said strand of double-stranded DNA nucleic acid, which starts outside the sequence of said at least one DNA tandem repeat and ends within the sequence of said at least one DNA tandem repeat, or conversely, which starts within the sequence of said at least one DNA tandem repeat and ends outside the sequence of said at least one DNA tandem repeat, wherein each of said first and second DNA-binding polypeptides is directly or indirectly linked to one endonuclease monomer or to one fragment of endonuclease monomer, wherein said fragment of endonuclease monomer still comprises the catalytic domain of said endonuclease monomer, and 
   wherein said first and second DNA-binding polypeptides induce a partial or complete deletion of said at least one DNA tandem repeat.   
     
     
         2 . The composition or kit of  claim 1 , wherein the DNA target site of said first DNA-binding polypeptide is a DNA target site as defined in  claim 1  i. and the DNA target site of said second DNA-binding polypeptide is a DNA target site as defined in  claim 1  ii. 
     
     
         3 . The composition or kit of  claim 1  or  2 , wherein the sequence of the DNA target site of said first DNA-binding polypeptide is different from the sequence of the DNA target site of said second DNA-binding polypeptide. 
     
     
         4 . The composition or kit of any one of  claims 1 - 3 , wherein the endonuclease monomer or endonuclease monomer fragment of said first DNA-binding polypeptide dimerizes with the endonuclease monomer or endonuclease monomer fragment of said second DNA-binding polypeptide, when said first and second DNA-binding polypeptides are bound to their respective DNA target sites. 
     
     
         5 . The composition or kit of any one of  claims 1 - 4 , wherein said dimeric endonuclease is the endonuclease FokI. 
     
     
         6 . The composition or kit of any one of  claims 1 - 5 , wherein the nucleotide spacer length between the DNA target site of said first DNA-binding polypeptide and the DNA target site of said second DNA-binding polypeptide is of 15-24 nucleotides. 
     
     
         7 . The composition or kit of any one of  claims 1 - 6 , wherein said first and second DNA-binding polypeptides induce a double-strand break specifically in said double-stranded DNA nucleic acid. 
     
     
         8 . The composition or kit of any one of  claims 1 - 7 , wherein each of said first and second DNA-binding polypeptides further comprises a Nuclear Localization Signal (NLS). 
     
     
         9 . The composition or kit of any one of  claims 1 - 8 , wherein none of said first and second DNA-binding polypeptides comprises the acidic transcriptional Activator Domain (AD) of a TAL effector. 
     
     
         10 . A composition or kit, which comprises a first nucleic acid and a second nucleic acid, wherein said first nucleic acid codes for a first DNA-binding polypeptide, wherein said second nucleic acid codes for a second DNA-binding polypeptide, wherein said first DNA-binding polypeptide is different from said second DNA-binding polypeptide, and wherein said first DNA-binding polypeptide and said second DNA-binding polypeptide are as defined in any one of  claims 1 - 9 . 
     
     
         11 . A composition or kit, which comprises:
 a first recombinant nucleic acid vector and a second recombinant nucleic acid vector, wherein said first recombinant nucleic acid vector codes for a first DNA-binding polypeptide, wherein said second recombinant nucleic acid vector codes for a second DNA-binding polypeptide, wherein said first DNA-binding polypeptide is different from said second DNA-binding polypeptide, and wherein said first DNA-binding polypeptide and said second DNA-binding polypeptide are as defined in any one of  claims 1 - 9 ; and/or comprising   a first lentiviral vector pseudotyped particle and a second lentiviral vector pseudotyped particle, wherein said first lentiviral vector pseudotyped particle codes for a first DNA-binding polypeptide, wherein said second lentiviral vector pseudotyped particle codes for a second DNA-binding polypeptide, wherein said first DNA-binding polypeptide is different from said second DNA-binding polypeptide, and wherein said first DNA-binding polypeptide and said second DNA-binding polypeptide are as defined in any one of  claims 1 - 9 .   
     
     
         12 . The composition or kit of any one of  claims 1 - 11 , which is for use in the treatment and/or palliation and/or prevention of said neurological and/or muscular and/or skeletal disorder or disease. 
     
     
         13 . The composition or kit for the use according to  claim 12 , wherein said first and second DNA-binding polypeptides induces a deletion of said at least one DNA tandem repeat to a length below the pathological threshold. 
     
     
         14 . The composition or kit for the use according to  claim 12  or  13 , wherein said neurological and/or muscular and/or skeletal disease or disorder is a trinucleotide, tetranucleotide or pentanucleotide disease or disorder. 
     
     
         15 . The composition or kit for the use according to any one of  claims 12 - 14 , wherein said DNA sequence unit of said at least one DNA tandem repeat is  5′ CTG 3′ ,  5′ TTG 3′ ,  5′ GTC 3′ ,  5′ CCTG 3′ ,  5′ ATTCT 3′  or  5′ AGAAT 3′ . 
     
     
         16 . The composition or kit for the use according to any one of  claims 12 - 15 , wherein the sequence of said at least one DNA tandem repeat forms a secondary structure, which is a hairpin, a triple helix or a tetraplex secondary structure. 
     
     
         17 . The composition or kit for the use according to any one of  claims 12 - 16 , wherein said neurological and/or muscular and/or skeletal disease or disorder is a trinucleotide, tetranucleotide or pentanucleotide disease or disorder. 
     
     
         18 . The composition or kit for the use according to any one of  claims 12 - 17 , wherein said neurological and/or muscular and/or skeletal disease or disorder is DM1, SCA8, SCA12, HDL2, SBMA, HD, DRPLA, SCA1, SCA2, SCA3, SCA6, SCA7, SCA17, PSACH, DM2 or SCA10. 
     
     
         19 . The composition or kit for the use according to any one of  claims 12 - 18 , wherein said gene involved in a neurological and/or muscular and/or skeletal disorder or disease is:
 a gene coding for DMPK, ATXN8, PPP2R2B, JPH3, AR, HTT, ATN1, ATXN1, ATXN2, ATXN3, CACNA1A, ATXN7, TBP, COMP, ZNF9 or ATXN10, or   the human gene coding for DMPK, ATXN8, PPP2R2B, JPH3, AR, HTT, ATN1, ATXN1, ATXN2, ATXN3, CACNA1A, ATXN7, TBP, COMP, ZNF9 or ATXN10,   and wherein said gene comprises said at least one DNA tandem repeat.   
     
     
         20 . The composition or kit for the use according to any one of  claims 12 - 19 , wherein said adjacent units of TAL effector tandem repeat comprise one or several copy(ies) of at least one sequence selected from the group consisting of SEQ ID NOs: 25, 26, 46 and 55, and/or comprise one or several copy(ies) of at least one of the sequences of TAL effector tandem repeat units of the DNA-binding polypeptide, which is coded by the plasmid deposited at the Collection Nationale de Culture de Microorganismes (C.N.C.M.), Paris, France, under deposit number I-4804 or under deposit number I-4805. 
     
     
         21 . The composition or kit for the use according to any one of  claims 12 - 20 , wherein said DNA target site of  claim 1  i. is the sequence of SEQ ID NO: 10 or 11, and/or wherein said DNA target site of  claim 1  ii. is the sequence of SEQ ID NO: 4 or 5, and/or wherein said first and second DNA-binding polypeptides are the polypeptides coded by the sequences of SEQ ID NOs: 1 and 2, respectively. 
     
     
         22 . A method for producing a product that is useful for fully or partially deleting a DNA tandem repeat that is contained in a double stranded DNA, wherein said DNA tandem repeat forms, in said double stranded DNA, a secondary structure, which is a hairpin, a triple helix or a tetraplex structure, wherein said method comprises producing a pair of DNA-binding polypeptides, wherein said pair of DNA-binding polypeptides is a first DNA-binding polypeptide and a second DNA-binding polypeptide as defined in any one of  claims 1 - 9 , and wherein said pair of DNA-binding polypeptides is a product useful for said full or partial DNA tandem repeat deletion. 
     
     
         23 . A method for fully or partially deleting in vitro a DNA tandem repeat that is contained in a double stranded DNA, wherein said method comprises placing in vitro said double-stranded DNA into contact with:
 a first DNA-binding polypeptide and a second DNA-binding polypeptide as defined in any one of  claims 1 - 9 , wherein said first and second DNA-binding polypeptides induce the full or partial deletion of said DNA tandem repeat, or with   a first nucleic acid and a second nucleic acid as defined in  claim 10 , wherein the first and second DNA-binding polypeptides, which are coded by said first and second nucleic acids induce the full or partial deletion of said DNA tandem repeat, or with   a first recombinant nucleic acid vector and a second recombinant nucleic acid vector as defined in  claim 11 , wherein the first and second DNA-binding polypeptides, which are coded by said first and second recombinant nucleic acid vectors, induce the full or partial deletion of said DNA tandem repeat, or with   a first lentiviral vector pseudotyped particle and a second lentiviral vector pseudotyped particle as defined in  claim 11 , wherein the first and second DNA-binding polypeptides, which are coded by said first and second lentiviral vector pseudotyped particles induce the full or partial deletion of said DNA tandem repeat.