Methods and products for increasing frataxin levels and uses thereof

Abstract

Methods and products (e.g., recombinant proteins) are described for increasing frataxin expression/levels in a cell, as well as uses of such methods and products, for example for the treatment of Friedreich ataxia in a subject suffering therefrom.

1 - 20 . (canceled) 
     
     
         21 . A method for treating Friedreich ataxia in a subject comprising administering to said subject a transcription activator-like (TAL) effector-based recombinant protein or a nucleic acid encoding said recombinant protein, said recombinant protein comprising:
 i) a TAL effector domain comprising a repeat variable domain (RVD) comprising at least 10 tandem repeat monomers and a half monomer at the C-terminal end of the RVD, each monomer corresponding to an HD, NG, NN, NI, NK or NS module consisting of the amino acid sequence set forth in SEQ ID NO: 65 and the half monomer being a truncated monomer consisting of the first 13 contiguous amino acids of SEQ ID NO: 65,   ii) a nuclear localization signal; and   iii) a transcription activation domain,   wherein said RVD binds to a sequence within nucleotides 5-50 or 81-119 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO: 88 of a mammalian frataxin gene comprising at least 75 repeats of GAA trinucleotides in intron 1 thereof, and wherein said recombinant protein increases frataxin expression in said cell.   
     
     
         22 . A method for increasing frataxin expression in a cell comprising a frataxin gene comprising at least 75 repeats of GAA trinucleotides in intron 1 thereof, the method comprising transducing said cell with a transcription activator-like (TAL) effector-based recombinant protein or nucleic acid encoding said recombinant protein, said recombinant protein comprising: i) a TAL effector domain comprising a repeat variable domain (RVD) comprising at least 10 tandem repeat monomers and a half monomer at the C-terminal end of the RVD, each monomer corresponding to an HD, NG, NN, NI, NK or NS module consisting of the amino acid sequence set forth in SEQ ID NO: 65 and the half monomer being a truncated monomer consisting of the first 13 contiguous amino acids of SEQ ID NO: 65,
 ii) a nuclear localization signal; and   iii) a transcription activation domain,   wherein said RVD binds to a sequence within nucleotides 5-50 or 81-119 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO: 88 of a mammalian frataxin gene in said cell, thereby increasing frataxin expression in said cell.   
     
     
         23 . The method of  claim 21 , wherein said RVD binds to a sequence within nucleotides 81-116 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO:88. 
     
     
         24 . The method of  claim 21 , wherein said RVD binds to:
 i) Nucleotides 5-18 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO: 88;   ii) Nucleotides 21-34 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO: 88;   iii) Nucleotides 24-37 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO: 88;   iv) Nucleotides 37-50 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO: 88;   v) Nucleotides 81-94 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO: 88;   vi) Nucleotides 92-105 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO: 88; or   vii) Nucleotides 103-116 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO: 88; or   viii) Nucleotides 106-119 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO: 88.   
     
     
         25 . The method of  claim 23 , wherein said RVD binds to:
 i) Nucleotides 81-94 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO: 88;   ii) Nucleotides 92-105 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO: 88; or   iii) Nucleotides 103-116 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO: 88.   
     
     
         26 . The method of  claim 21 , wherein each of said repeat monomers corresponds to an HD, NG, NN or NI module and wherein said RVD comprises the following configuration of modules:
 i) HD-HD-HD-NG-NG-NN-NN-NN-NG-HD-NI-NG-NN;   ii) HD-HD-NG-NN-NN-NG-NG-NN-HD-NI-HD-NG-HD;   iii) NN-NN-NG-NG-NN-HD-NI-HD-NG-ND-HD-NN-NG;   iv) NN-HD-NG-NG-NG-NN-HD-NI-HD-NI-NI-NI-NN;   v) NI-NN-NG-NN-HD-NG-NI-NI-NN-HD-NG-NN;   vi) NN-NN-NN-NI-NI-NN-NG-NG-HD-NG-NG-HD-HD;   vii) HD-HD-NG-NN-NI-NN-NN-NG-HD-NG-NI-NI; or   viii) NN-NI-NN-NN-NG-HD-NG-NI-NI-HD-HD-NG;   wherein the C-terminal module in said configuration of modules corresponds to the half monomer,   wherein said HD module consists of the amino acid sequence of SEQ ID NO: 61; said NG module consists the amino acid sequence of SEQ ID NO: 59, said NI module consists of the amino acid sequence of SEQ ID NO: 57, and said NN module consists of the amino acid sequence of SEQ ID NO: 63, and   wherein said RVD binds to a sequence within nucleotides 5-50 or 81-119 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO: 88.   
     
     
         27 . The method of  claim 23 , wherein each of said repeat monomers corresponds to an HD, NG, NN or NI module and wherein said RVD comprises the following configuration of modules:
 i) NI-NN-NG-NN-HD-NG-NI-NI-NN-HD-NG-NN;   ii) NN-NN-NN-NI-NI-NN-NG-NG-HD-NG-NG-HD-HD; or   iii) HD-HD-NG-NN-NI-NN-NN-NG-HD-NG-NI-NI.   wherein the C-terminal module in said configuration of modules corresponds to the half monomer,   wherein said HD module consists of the amino acid sequence of SEQ ID NO: 61; said NG module consists the amino acid sequence of SEQ ID NO: 59, said NI module consists of the amino acid sequence of SEQ ID NO: 57, and said NN module consists of the amino acid sequence of SEQ ID NO: 63; and   wherein said RVD binds to a sequence within nucleotides 81-116 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO: 88.   
     
     
         28 . The method of  claim 21 , wherein said transcription activation domain comprises at least one VP16 synthetic transcription activation domain. 
     
     
         29 . The method of  claim 21 , wherein said transcription activation domain comprises at least one VP64 synthetic transcription activation domain. 
     
     
         30 . The method of  claim 21 , wherein said nuclear localization signal is a mammalian nuclear localization signal derived from the simian virus 40 large T antigen. 
     
     
         31 . The method of  claim 22 , wherein said RVD binds to a sequence within nucleotides 81-116 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO:88. 
     
     
         32 . The method of  claim 22 , wherein said RVD binds to:
 i) Nucleotides 5-18 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO:88;   ii) Nucleotides 21-34 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO:88;   iii) Nucleotides 24-37 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO:88;   iv) Nucleotides 37-50 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO:88;   v) Nucleotides 81-94 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO:88;   vi) Nucleotides 92-105 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO:88; or   vii) Nucleotides 103-116 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO:88; or   viii) Nucleotides 106-119 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO:88.   
     
     
         33 . The method of  claim 31 , wherein said RVD binds to:
 i) Nucleotides 81-94 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO:88;   ii) Nucleotides 92-105 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO:88; or   iii) Nucleotides 103-116 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO:88.   
     
     
         34 . The method of  claim 22 , wherein each of said repeat monomers corresponds to an HD, NG, NN or NI module and wherein said RVD comprises the following configuration of modules:
 ix) HD-HD-HD-NG-NG-NN-NN-N N-NG-HD-NI-NG-NN;   x) HD-HD-NG-NN-NN-NG-NG-NN-HD-NI-HD-NG-HD;   xi) NN-NN-NG-NG-NN-HD-NI-HD-NG-ND-HD-NN-NG;   xii) NN-HD-NG-NG-NG-NN-HD-NI-HD-NI-NI-NI-NN;   xiii) NI-NN-NG-NN-HD-NG-NI-NI-NN-HD-NG-NN;   xiv) NN-NN-NN-NI-NI-NN-NG-NG-HD-NG-NG-HD-HD;   xv) HD-HD-NG-NN-NI-NN-NN-NG-HD-NG-NI-NI; or   xvi) NN-NI-NN-NN-NG-HD-NG-NI-NI-HD-HD-NG;   wherein the C-terminal module in said configuration of modules corresponds to the half monomer,   wherein said HD module consists of the amino acid sequence of SEQ ID NO: 61; said NG module consists the amino acid sequence of SEQ ID NO: 59, said NI module consists of the amino acid sequence of SEQ ID NO: 57, and said NN module consists of the amino acid sequence of SEQ ID NO: 63, and   wherein said RVD binds to a sequence within nucleotides 5-50 or 81-119 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO:88.   
     
     
         35 . The method of  claim 31 , wherein each of said repeat monomers corresponds to an HD, NG, NN or NI module and wherein said RVD comprises the following configuration of modules:
 i) NI-NN-NG-NN-HD-NG-NI-NI-NN-HD-NG-NN;   ii) NN-NN-NN-NI-NI-NN-NG-NG-HD-NG-NG-HD-HD; or   iii) HD-HD-NG-NN-NI-NN-NN-NG-HD-NG-NI-NI.   wherein the C-terminal module in said configuration of modules corresponds to the half monomer,   wherein said HD module consists of the amino acid sequence of SEQ ID NO: 61; said NG module consists the amino acid sequence of SEQ ID NO: 59, said NI module consists of the amino acid sequence of SEQ ID NO: 57, and said NN module consists of the amino acid sequence of SEQ ID NO: 63; and   wherein said RVD binds to a sequence within nucleotides 81-116 of the frataxin promoter nucleotide sequence set forth in SEQ ID NO:88.   
     
     
         36 . The method of  claim 22 , wherein said transcription activation domain comprises at least one VP16 synthetic transcription activation domain. 
     
     
         37 . The method of  claim 22 , wherein said transcription activation domain comprises at least one VP64 synthetic transcription activation domain. 
     
     
         38 . The method of  claim 22 , wherein said nuclear localization signal is a mammalian nuclear localization signal derived from the simian virus 40 large T antigen.