Patent Publication Number: US-10323073-B2

Title: CRISPR-based methods and products for increasing frataxin levels and uses thereof

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     The present application is a National Stage Application of PCT Application No. PCT/CA2015/050213 filed on Mar. 20, 2015, and published in English under PCT Article 21(2), which claims the benefit of U.S. Provisional Application Ser. No. 61/955,882 filed on Mar. 20, 2014. All documents above are incorporated herein by reference in their entirety. 
    
    
     SEQUENCE LISTING 
     Pursuant to 37 C.F.R. 1.821(c), a sequence listing is submitted herewith as an ASCII compliant text file named “11229_355_SeqList_v2.txt”, created Nov. 21, 2018 and having a size of about 209 KB, which is incorporated herein by reference. 
     FIELD OF THE INVENTION 
     The present invention relates to increasing frataxin expression and/or levels and uses thereof, for example for the treatment of Friedreich ataxia. 
     BACKGROUND OF THE INVENTION 
     Frataxin 
     Frataxin is a small protein (Isoform 1: NCBI NM_000144.4, NP_000135 (210 aa), SEQ ID NO: 1; Isoform 2: NM_181425, NP_852090 (196 aa), SEQ ID NO: 2; Isoform 3: NM_001161706, NP_001155178, (171 aa), SEQ ID NO: 3; Uniprot Q16595; ENTREZ 2395; Ensembl ENSG00000165060; OMIM: 606829) found in cells throughout the body, with the highest levels in tissues with a high metabolic rate including heart, neurons, spinal cord, liver, pancreas (Langerhans cells), and muscles used for voluntary movement (skeletal muscles). Within cells, frataxin is found in mitochondria. It promotes the biosynthesis of heme as well as the assembly and repair of iron-sulfur clusters by delivering Fe2+ to proteins involved in these pathways. It also plays a primary role in the protection against oxidative stress through its ability to catalyze the oxidation of Fe2+ to Fe3+ and to store large amounts of the metal in the form of a ferrihydrite mineral. It is processed in two steps by mitochondrial processing peptidase (MPP). MPP first cleaves the precursor to intermediate form and subsequently converts the intermediate to a mature protein. Thus, in cells, 3 forms exist. For isoform 1, these forms are frataxin (56-210); frataxin (78-210) and frataxin (81-210), which is the main form of mature frataxin (Schoenfeld et al. 2005). 
     Friedreich Ataxia 
     Friedreich ataxia (FRDA), an autosomal recessive neurodegenerative and cardiac disease, is caused by a trinucleotide repeat expansion mutation in the first intron of the frataxin gene (FXN), which is located on the long arm of chromosome 9 (Location UCSC: 71,650,175 to 71715094 (64,920 bp; 9q21.11)). The mutation leads to a reduced expression of the frataxin mRNA and protein. Frataxin is essential for proper functioning of mitochondria. As noted above, it is involved in the removal of iron and when frataxin is reduced, iron builds up and causes free radical damage. Nerve and muscle cells are particularly sensitive to these deleterious effects. FRDA occurs in 1 in 50,000 persons in European populations but is much more frequent in the province of Quebec in Canada, because of founder effects. Males and females are affected equally. In the classic form, FRDA symptoms appear during or before the second decade of life. It is characterized by ataxia, areflexia, loss of vibratory sense and proprioception and dysarthria (Pandolfo 2012). Moreover, FRDA patients often have systemic involvement, with cardiomyopathy, diabetes mellitus and scoliosis. Early death can result from cardiomyopathy or associated arrhythmias. Degeneration of the dorsal root ganglion cells, their ascending dorsal spinal columns and the spinocerebellar tracts results in a progressive sensory ataxia. Many patients are wheelchair bound by their third decade. Associated oculomotor problems include optic atrophy, square-wave jerks and difficulty with fixation. Importantly, cognitive abilities are relatively spared. However, many patients suffer from depression. 
     The mutation responsible for FRDA is an unstable hyper-expansion of a GAA triplet repeat located in the first intron of the frataxin gene (Campuzano et al. 1996). In normal subjects, there are 6-34 repeats, whereas in FRDA patients there are 150 or more repeats. Patients with fewer repeats (150-200) have milder symptoms than those with longer sections with more triplet repeats (350 to 650). In some severely affected patients there are up to 1700 repeats. Since the frataxin gene mutation is located in an intron, it does not alter the amino acid sequence of the frataxin protein. There are 2-3% of FRDA patients who have a point mutation, either a missense or a non-sense. Some patients with a missense mutation have less severe symptoms because the mutated protein in still functional. 
     Pathological Mechanism 
     The pathological mechanisms have been reviewed by Pandolfo et al. (Pandolfo 2012). The repeated GAA triplets cause modifications in DNA methylation and the formation of triplex in the DNA, i.e., unusual non-B DNA conformations, which decrease transcription and subsequently reduce levels of the encoded protein, frataxin (level of expression is 5 to 35% of normal; (Coppola et al. 2006, Coppola et al. 2009)). Iron accumulation in mitochondria is mainly observed in the cardiac cells of patients and in the dentate nucleus of the brain. It is associated with oxidative damage. The reduction of frataxin leads to changes in gene expression of 185 different genes (Coppola et al. 2006, Coppola et al. 2009). Thus the reduction of frataxin has profound effects on several metabolic pathways and the correction of only one of these pathways by a drug may not be ideal. 
     Several strategies have been developed for treating Friedreich ataxia. These fall generally into the following 5 categories: 1) use of antioxidants to reduce the oxidative stress caused by iron accumulation in the mitochondria; 2) use of iron chelators to remove iron from the mitochondria; 3) use of Histone Deacetylase Inhibitors (HDACIs) to prevent DNA condensation and permit higher expression of frataxin; 4) use of molecules such as cisplatin, 3-nitroproprionnic acid (3-NP), Pentamidine or erythropoietin (EPO) to boost frataxin expression; and 5) gene therapy 
     The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety. 
     SUMMARY OF THE INVENTION 
     The present inventors have shown for the first time that frataxin expression can be directly and efficiently increased in cells from Friedreich Ataxia patients comprising a mutated frataxin gene using a modified CRISPR system. The method comprises delivering a protein comprising a transcription activation domain (TAD) to the endogenous frataxin promoter. Surprisingly, frataxin expression could be substantially increased without correcting the endogenous gene (i.e, removing excess trinucleotide repeats) or its methylation level. 
     Thus, the present invention relates to inducing or increasing frataxin expression/levels in a cell using a modified Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system/frataxin targeting system, and uses thereof. In an aspect, a “guide RNA” (gRNA) may be designed and used to specifically target the frataxin promoter to increase frataxin expression. 
     In a particular aspect, the present invention provides a modified CRISPR/Cas9-based system/frataxin targeting system comprising (or consisting of) a dCas9 fusion protein comprising two heterologous polypeptide domains and at least one gRNA to target the frataxin promoter. The first polypeptide domain comprises an inactive CRISPR-associated nuclease protein (dCas, such as dCas9) and the second polypeptide domain has transcription activation activity. This mutated inactive Cas nuclease (dCas, such as dCas9) fused with at least one transcription activation domain (TAD) is used to make a complex with the frataxin promoter and the gRNA targeting a nucleotide sequence within that promoter thereby enabling an increase in frataxin expression. 
     In a further aspect, the present invention provides a modified CRISPR/Cas9-based system/frataxin targeting system comprising (or consisting of) a first fusion protein comprising two heterologous polypeptide domains and at least one gRNA to target the frataxin promoter. The first polypeptide domain comprises a mutated inactive CRISPR-associated nuclease protein (dCas, such as dCas9) and the second polypeptide domain comprises a polypeptide sequence (i.e, a TAG) comprising a peptide epitope (in an embodiment, at least two peptide epitopes). The system further comprises a second fusion protein also comprising two heterologous polypeptide domains. The first polypeptide domain of the second fusion protein comprises an antigen binding domain (e.g., comprising an antigen-binding domain of an antibody, such as antibody scFv fragment) which is capable of binding to the peptide epitope of the first fusion protein (dCas/TAG), and the second polypeptide domain of the second fusion protein has transcription activation activity (e.g., it comprises at least one TAD). In this system, the first fusion protein may in embodiments comprise multiple repetitions of a peptide epitope (e.g., GCN4 peptide, SEQ ID NO: 4) fused to dCas9. This peptide epitope is recognized by the binding domain of the second fusion protein (such as an antibody/antibody fragment), which is fused to a transcription activation domain (e.g., VP64). The presence of multiple repetitions of the peptide epitopes on the first fusion protein comprising dCas (e.g., dCas9) allows recruitment of multiple second fusion proteins comprising transcription activators (comprising TADs) at a single target site on the frataxin promoter, thereby further increasing its expression. (see  FIG. 13 , from Tanenbaum et al., Cell, 2014); 
     In another aspect, the present invention provides a modified CRISPR/Cas9-based system/frataxin targeting system comprising (or consisting of) an MS2-fusion protein to be used in conjunction with a modified gRNA (comprising one or more (e.g., two) MS2 sequences which bind the MS2 polypeptide) and a dCas9-comprising fusion protein having transcription activating activity (comprising one or more TADS). The MS2 fusion protein comprises an MS2 domain (e.g., SEQ ID NO: 7, which recognizes specific MS2 sequences in the modified gRNA) and at least one (in an embodiment at least two) TAD (see for example  FIG. 15 ). The MS2 fusion protein may comprises an MS2 polypeptide (SEQ ID NO: 7;  FIG. 15B ), the p65 polypeptide (SEQ ID NO: 8,  FIG. 15C ) and the HSF1 (human Heat-shock factor 1) polypeptide (SEQ ID NO: 9,  FIG. 15D ). 
     Accordingly, in an aspect, the present invention also provides at least one guide RNA (gRNA) comprising i) a gRNA guide sequence of at least 10 contiguous nucleotides corresponding to a target sequence in a frataxin promoter polynucleotide sequence; and ii) a Cas9 recognition sequence, wherein the target sequence is contiguous to a protospacer adjacent motif (PAM) recognized by a ribonucleoprotein complex comprising a Cas9 protein and/or an inactive Cas9 (dCas9) protein lacking nuclease activity. 
     In an further aspect, the present invention provides a frataxin targeting system comprising: a. a1) at least one gRNA (in a further embodiment, one) as; or a2) a vector comprising a nucleic acid sequence corresponding to the at least one gRNA and for expressing the at least one gRNA; and b. b1) a dCas9 fusion protein comprising an inactive Cas9 (dCas9) polypeptide domain lacking nuclease activity, wherein the dCas9 fusion protein further comprises i) at least one nuclear localization signal (in a further embodiment, one); and/or ii) (a) at least one transcription activation domain and/or (b) a polypeptide domain comprising at least one peptide epitope (in a further embodiment, one); or b2) a vector comprising a nucleic acid sequence encoding the fusion protein, for expression of the fusion protein. 
     In embodiments, the dCas9 protein or dCas9 polypeptide domain is derived from  Streptococcus pyogenes, Streptococcus thermophilus  or  Neisseria meningitides . In an embodiment, the dCas9 protein or the dCas9 polypeptide domain is derived from  Streptococcus pyogenes.    
     In an embodiment, the PAM is a NGG trinucleotide-sequence. 
     In an embodiment the dCas9 protein or dCas9 polypeptide domain comprises a mutation at an aspartic acid corresponding to position 10 of a Cas9 amino acid sequence as set forth in SEQ ID NO: 35 and/or at a histidine corresponding to position 840 of a Cas9 amino acid sequence as set forth in SEQ ID NO: 35. In an embodiment, the dCas9 protein or dCas9 polypeptide domain comprises mutations corresponding to D10A and H840A. 
     In an embodiment, the gRNA guide sequence consists of 12-22 (in embodiments, 10-20, 10-19, 10-18, 18-22, 18-20, 18-19, 19-22, or 20-22) and preferably 18, 19 or 20 nucleotides targeting a particular target region. In embodiments, the gRNA guide sequence is perfectly identical to a target region. The at least one gRNA may target the promoter region of the frataxin gene or an enhancer region of the frataxin gene. 
     In an embodiment the 3′ end of the target sequence of the gRNA in the frataxin promoter ends at nucleotide position i) 4264; ii) 4670, iii) 4859; iv) 5023; or v) 5107, wherein the nucleotide position corresponds to that of the frataxin polynucleotide gene sequence having NCBI reference number NG_008845 (SEQ ID NO: 87). 
     In an embodiment the 3′ end of the target sequence of the gRNA in the frataxin promoter ends at nucleotide position i) 4670; ii) 4701; iii) 4742; ii) 4859; or iii) 5107, wherein the nucleotide position corresponds to that of the frataxin polynucleotide gene sequence having NCBI reference number NG_008845 (SEQ ID NO: 87). 
     In an embodiment the target sequence of the gRNA in the frataxin promoter ends at wherein the 3′ end of the target sequence ends at nucleotide position ii) 4670, ii) 4859; or iii) 5107, wherein the nucleotide positions corresponds to that of the frataxin polynucleotide gene sequence having NCBI reference number NG_008845 (SEQ ID NO: 87). 
     In an embodiment the target sequence of the gRNA in the frataxin promoter is comprised within a nucleotide sequence corresponding to i) nucleotides 3961-4258; ii) nucleotides 4268-49513; iii) nucleotides 4268-4996; iv) nucleotides 5027-5150; v) nucleotides 4268-4595; vi) nucleotides 4621-4996; vii) nucleotides 4561-4996; or viii) nucleotides 4280-5000; iv) the complement of anyone of i) to viii), wherein the nucleotide positions corresponds to that of the frataxin polynucleotide gene sequence having NCBI reference number NG_008845 (SEQ ID NO: 87). 
     In an embodiment, the RNA guide sequence and the target sequence do not comprise more than one NGG sequence. In an embodiment the RNA guide sequence and the target sequence do not comprise an NGG sequence. 
     In an embodiment, the target sequence of the gRNA in the frataxin promoter comprises or consist of: 
     In an embodiment, the target sequence of the gRNA in the frataxin promoter comprises or consists of the nucleotide sequence of: 
     i) Positions 4246-4264; 
     ii) Positions 4652-4670; 
     iii) Positions 4841-4859; 
     iv) Positions 5005-5023; or 
     v) Positions 5089-5107,
         of the frataxin polynucleotide gene sequence having NCBI reference number NG_008845 (SEQ ID NO: 87, see also  FIG. 2 ).       

     In an embodiment, the Cas9 recognition sequence comprises or consists of the sequence as set forth in SEQ ID NO: 67. In an embodiment, the gRNA comprises or consists of a sequence as set forth in any one of SEQ ID NOs: 57-61. 
     In an embodiment, the above dCas9 fusion protein comprises a polypeptide domain comprising at least one peptide epitope, and the system further comprises a fusion protein comprising i) an antigen-binding region which binds to the at least one peptide epitope and ii) at least one transcription activation domain. In an embodiment, the antigen-binding region comprises a single chain variable fragment which binds to the at least one peptide epitope. In an embodiment the dCas9 fusion protein comprises a polypeptide domain comprising at least two peptide epitopes. 
     In an embodiment the present invention further provides i) an MS2 fusion protein comprising a) at least one nuclear localization signal and/or b. b1) an MS2 polypeptide and b2) at least one transcription activation domain, or ii) a vector comprising a nucleic acid sequence encoding the MS2 fusion protein. In an aspect, the MS2 fusion protein is used with a gRNA which comprises an MS2 nucleic acid sequence in a tetraloop and an MS2 nucleic acid sequence in a stem loop of the gRNA which is capable of binding to the MS2 polypeptide. 
     Accordingly, the above gRNA may further comprise at least one MS2 nucleic acid sequence. In an embodiment, the gRNA comprises two MS2 sequences, one in a tetraloop one in a stem loop of the gRNA. In an embodiment, the MS2 nucleic acid sequence comprises or consists of the sequence set forth in SEQ ID NO: 68. In an embodiment, the gRNA comprises or consists of a sequence as set forth in any one of SEQ ID NOs: 62-66. 
     In an embodiment, the above transcription activation domain comprise a VP16 (SEQ ID NO: 17), VP64 or VP160 (SEQ ID NO: 18) transcription activation domain. In another embodiment, transcription activation domain comprises one or multiple copies of a VP16 TAD (SEQ ID NO: 17). In embodiments, the transcription activation domain may comprise a HSF1 (SEQ ID NO: 9) or a p65 activation (SEQ ID NO: 8) domain. The fusion protein may further comprise a linker connecting the various heterologous domains of the fusion protein (e.g., between the dCas9 polypeptide domain and the TAD or peptide epitope; between the MS2 polypeptide and the TAD; between peptide epitopes or between twoTADs). The fusion proteins of the present invention preferably comprise at least one nuclear localization signal. In an embodiment, the fusion proteins of the present invention comprise two NLSs. In an embodiment, the NLS is a mammalian nuclear localization signal derived from the simian virus 40 large T antigen. In a particular embodiment, the NLS comprises the sequence PKKKRKV (SEQ ID NO: 12). In a particular embodiment, the transcription activation domain comprises 10 copies of a VP16 TAD (VP160) (SEQ ID NO: 18). 
     In an embodiment, the above inactive dCas9 fusion protein has an amino acid sequence at least 95% (in embodiments at least 96,%, 97%, 98% or 99%) identical to the sequence set forth in  FIG. 3  (SEQ ID NO: 33). 
     In embodiments the dCas9 fusion protein comprises between 2 and 24 (in an embodiment 10) GCN4 peptide epitopes. In an embodiment, the dCas9 fusion protein comprises a sunTAG polypeptide sequence as set forth in SEQ ID NO: 5 or 6. 
     In another embodiment, the amino acid sequence of the dCAs/TAD protein of the present invention comprises an amino acid sequence at least 95% identical to amino acids 10 to 1538 of the sequence set forth in  FIG. 3  (SEQ ID NO: 34). In another embodiment, the amino acid sequence of the dCAs/TAD protein of the present invention comprises an amino acid sequence at least 95% identical to amino acids 10 to 1535 of the sequence set forth in  FIG. 3  (amino acid 1-1526 of SEQ ID NO: 34). In particular embodiments, the dCas/TAD protein of the present invention comprises or consists of a sequence at least 95% (i.e. 95%, 96%, 97%, 98%, 99%, 99.5% or 100%) identical to the above-mentioned amino acid sequences of  FIG. 3  (SEQ ID NOs:32 and 34). 
     In a further embodiment, the above-mentioned inactive fusion protein further comprises a protein transduction domain (PTD) to target the protein into a cell. In a particular embodiment, the PTD is TAT or Pep-1. In an embodiment, the TAT-PTD comprises the sequence SGYGRKKRRQRRRC (SEQ ID NO: 36). 
     In a further aspect, the fusion proteins and/or gRNAs of the present invention may be complexed with liposomes to facilitate their entry into target cells. Liposomes may be used alone or in combination with a PTD domain. 
     The present invention also provides an isolated polynucleotide encoding one or more of the above-mentioned gRNAs alone or in combination with the above-mentioned inactive fusion protein of the present invention. Vectors comprising one or more of such polynucleotides are also provided. A vector may encode for several gRNAs or fusion proteins. In a particular embodiment, the vector comprises a polynucleotide sequence at least 95% (i.e., 95%, 96%, 97%, 98%, or 99%) identical to the polynucleotide sequence as set forth in  FIG. 4  (SEQ ID NO: 55), or to any one of SEQ ID NOs: 93-100. 
     In a particular embodiment, the vector of the present invention is a viral vector. In a particular embodiment, the viral vector is derived from a retrovirus, a lentivirus, an adeno associated virus, an adenovirus or a Herpes virus. In an embodiment, the vector is a viral vector. In an embodiment, the vector is an AAVDJ-8 or AAV2DJ9 adeno-associated virus vector. 
     In another aspect, the present invention also concerns a composition or combination comprising at least one of: 
     i) the above-mentioned vectors; or 
     ii) one or more gRNAs of the present invention; 
     iii) the above frataxin targeting system; and 
     iv) a cells comprising any one of i) to iii)
         The composition may further comprise a physiologically acceptable (e.g., a pharmaceutically acceptable) carrier.       

     In a further aspect, the present invention also concerns a kit comprising at least one of: 
     i) the above-mentioned vectors; or 
     ii) one or more of the above gRNAs; 
     iii) the above frataxin targeting system; and 
     iv) a cell comprising any one of i) to iii) 
     The present invention also provides a cell comprising one or more of the above-mentioned gRNAs, fusion proteins, frataxin targeting system, combinations, isolated polynucleotides and/or vectors of the present invention. 
     The present invention further relates to one or more of the above-mentioned gRNAs, frataxin targeting system, isolated polynucleotides, vectors, cells, compositions, combinations or kit for increasing frataxin expression in a cell. 
     In a related aspect, the present invention relates to a use of one or more of the above-mentioned gRNAs, frataxin targeting system, isolated polynucleotides, vectors, cells, compositions, combinations or kit for increasing frataxin expression in a cell. 
     In embodiments, the cell does not express frataxin or the cell expresses a low level of frataxin as compared to a normal cell. In an embodiment, the cell has an abnormal number of GAA trinucleotide repeats in intron 1 of the frataxin gene. In a particular embodiment the cell comprises at least 35, preferably at least 80, more preferably at least 150 and even more preferably at least 250 or more GAA trinucleotide repeats. In an embodiment, the cell is from a subject suffering from FRDA. 
     The present invention further relates to one or more of the above-mentioned gRNAs, frataxin targeting system, isolated polynucleotides, vectors, cells, compositions, combinations or kit for the treatment of Friedreich ataxia. 
     The present invention also relates to a use of one or more the above-mentioned gRNAs, frataxin targeting system, isolated polynucleotides, vectors, cells, compositions, combinations or kit for treating Friedreich ataxia. 
     The present invention relates to one or more the above-mentioned gRNAs, frataxin targeting system, isolated polynucleotides, vectors, cells, compositions, kits or combination for the preparation of a medicament for treating Friedreich ataxia. 
     In a related aspect, the present invention relates to a use of one or more the above-mentioned gRNAs, frataxin targeting system, isolated polynucleotides, vectors, cells, compositions, kits or combinations for the preparation of a medicament for treating Friedreich ataxia. 
     The present invention also relates to a method of increasing frataxin expression in a cell comprising expressing at least one of the above-mentioned gRNAs, or frataxin targeting systems, or administering at least one of the above-mentioned frataxin targeting systems, vectors, cells, compositions or combinations. 
     The present invention further concerns a method for treating Friedreich ataxia in a subject comprising expressing at least one the above-mentioned gRNAs, polynucleotides, vectors or frataxin targeting systems in a cell of the subject. 
     The present invention further concerns a method for treating Friedreich ataxia in a subject comprising administering to the subject one or more of the above-mentioned gRNAs frataxin targeting systems, vectors cells, compositions, or combinations. 
     In an embodiment, the above-mentioned method comprises transducing one or more viral vectors for expressing a gRNA and/or a Cas/TAD protein of the present invention in the cell. 
     Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a schematic representation of the single-guide RNA (gRNA; SEQ ID NO: 110) and Cas9 nuclease protein targeting a sequence (sense, SEQ ID NO: 112; antisense, SEQ ID NO: 111) on a chromosome (Mali et al. 2013b); 
         FIG. 2  shows the partial sequence of the human frataxin promoter (SEQ ID NO: 11, LOCUSNG_00884571616bpDNAlinearPRI02-JUL-2013, human frataxin gene ACCESSION NG_008845 VERSION NG_008845.2G1:254826725). Potential protospacer adjacent motifs (PAM) along the shown portion of the frataxin promoter are shaded (ngg sequences). The first positions of the transcriptional start sites (agt and cac) positioned at 5001 and 5159 are boxed. Start codon (ATG) of the protein coding sequence at position 5221-5223 is in bold capital letters. Primers used to amplify the frataxin promoter are shaded (4063-4086 [SEQ ID NO: 113] and 5454-5362 [SEQ ID NO: 114]). Exemplary target sequences (4246-4264 [SEQ ID NO: 92]; 4652-4670 [SEQ ID NO: 91]; 4841-4859 [SEQ ID NO: 90]; 5005-5023 [SEQ ID NO: 89]; 5089-5107 [SEQ ID NO: 88]) on the frataxin promoter, according to an embodiment of the present invention, are in bold and underlined and are also presented in Table 4 in Example 1; 
         FIG. 3  shows the amino acid sequence of dCas9-VP160 (SEQ ID NO: 32) comprising two Nuclear Localization Signal (NLS) (underlined and bold—PKKKRKV, SEQ ID NO: 12) at the N-Terminal and C-Terminal ends of dCas9-VP160. The sequence of the ten VP16 Transcription Activation Domain TAD (VP160) at the C-terminal of dCas9-VP160 are underlined (VP160, SEQ ID NO: 18; VP16, SEQ ID NO: 17). The theoretical isoelectric point of the dCas9 protein is 5.89 and its molecular weight is 176880.44 Daltons; 
         FIG. 4  shows the nucleic acid sequence of the pAC154-dual-dCas9-VP160 plasmid (SEQ ID NO: 55) for expression of the gRNA A and of the dCas9-VP160. Underlined and in bold is the sequence targeted in the promoter of the human frataxin gene by an exemplary gRNA (gRNA A) of the present invention (SEQ ID NO: 88); 
         FIG. 5  shows a partial sequence of the pAC154 plasmid (sense strand, SEQ ID NO: 115; antisense strand, SEQ ID NO: 116; full amino acid sequence, SEQ ID NO: 117) with the corresponding amino acid sequences coding for VP160 (SEQ ID NO: 18), composed of ten VP16 sequences. Each VP16 sequence (SEQ ID NO: 17) is shaded; 
         FIG. 6  shows that gRNAs of the present invention can efficiently bind to their target sequence on the frataxin promoter. Results are from a surveyor enzyme test for lysates obtained from 293T cells transfected with an active Cas9 nuclease and each of the exemplary gRNAs (named A, B, C, D and E) targeting the promoter of the human Frataxin gene (see  FIG. 2  and SEQ ID NO: 11). The control lane corresponds to untransfected cells. When the gRNA binds to its target, the Cas9 nuclease forms a complex that cuts the DNA. This DNA break is spontaneously repaired by Non Homologous End Joining, which results in micro-insertions or micro-deletion (INDELs) in the DNA. These INDELs are detected by PCR amplification followed by digestion with the Surveyor enzyme that cuts the mismatched DNA double strands. Results show that gRNAs A, C and D can bind to their respective target sequence in the frataxin promoter. However, gRNAs B and E did not effectively bind to their respective target sequence and thus no additional DNA band was detected following the Surveyor enzyme test; 
         FIG. 7  shows that HA-dCas9-VP160 protein is efficiently expressed in cells. Results shown are from a Western blot using an HA antibody on lysates of 293T cells transfected with the HA-dcas9-VP160 gene and gRNAs A, B, C, D and E; 
         FIG. 8  shows a schematic representation of a single guide RNA (gRNA) that attaches to the promoter of the frataxin gene and forms a complex with the inactivated Cas9 (dCas9) coupled with VP160. This complex recruits transcription factors leading to the activation of frataxin gene transcription; 
         FIG. 9  shows that gRNAs of the present invention increase the expression of the frataxin gene in cells. Quantitative frataxin expression levels were determined by qRT-PCR on mRNA of 293T cells transfected with a plasmid coding for dCas9-VP160 and for one (or several) gRNA(s) targeting the promoter of the human frataxin gene. gRNAs A, C and D and various combinations of gRNAs increased the expression of the frataxin mRNA; 
         FIG. 10  shows that frataxin protein expression is increased by the presence of gRNAs. The top panel shows a Western blot for the frataxin and the beta-actin proteins using a lysate from 293T cells treated with dCas9-VP160 and one or combinations of several gRNAs targeting the promoter of the human frataxin gene. The intensity of the frataxin band was normalized with the intensity of the beta-actin band from the same sample. The bottom panel shows the average results for 3 Western blots. The normalized intensity of the frataxin protein was increased by the presence of the gRNAs used alone or in various combinations; 
         FIG. 11  shows that gRNAs targeting the frataxin promoter can increase the expression of frataxin mRNA in Friedreich cells. Results are from GFPqRT-PCR for the Frataxin mRNA from lysate of Friedreich cells 60 hours after nucleofection of a plasmid coding for dCas9-VP64 and one or several gRNA targeting the promoter of the human frataxin gene. Results were normalized with cells transfected with GFP and also normalized relative to 2 different control RNAs (left columns: ↓GAPDH; right columns: 18S rRNA); 
         FIG. 12  shows that gRNAs targeting the frataxin promoter can increase the expression of frataxin protein in Friedreich cells. Top panel: Western blot of frataxin protein expression 60 hours following nucleofection of the plasmid coding for gRNA and the dCas9-VP64 in Friedreich fibroblasts. Bottom panel: Frataxin expression was quantified and normalized with β-actin expression (the frataxin antibody was from Mitosciences and the β-actin antibody was purchased from Sigma Aldrich); 
         FIG. 13  shows an alternative embodiment of the methods of the present invention using a 3-component modified CRISPR system i.e., a gRNA; a modified dCas9 and a protein having antigen-binding activity (scFv fragment) coupled to a transcription activation domain. In this method, multiple repetitions of a peptide epitope (e.g., GCN4 peptide, SEQ ID NO: 4) are fused to dCas9. This peptide epitope is recognized by an antibody/antibody fragment coupled to a transcription activation domain (e.g., VP64). The presence of multiple repetitions of the peptide epitopes on the dCas9 fusion protein allows recruitment of multiple transcription activation domains (TADs) at a single target site on the frataxin promoter, thereby further increasing its expression. (Figure from Tanenbaum et al., Cell, 2014); 
         FIG. 14  shows an alternative embodiment of the methods of the present invention using a 3-component system i.e., a modified gRNA, a dCas9-TAD fusion protein and a MS2-p65-HSF1 fusion protein (SEQ ID NO: 10). Two MS2 nucleotide sequences were added: one on the tetraloop and one on the stem loop of the gRNA. A fusion protein comprising the MS2 peptide (SEQ ID NO: 7) and two TADs (e.g., the p65 (SEQ ID NO: 8) and HSF1 (SEQ ID NO: 9) transcription activation domains (TADs)) binds to each of the MS2 nucleotide sequences included in the gRNAs. The presence of 3 TADs (e.g., VP64 on the dCas9 protein and p65 and HSF1) produces a strong synergistic effect on the transcription of the gene (frataxin) targeted by the gRNA. (Figure from Konermann et al., Nature, 2014); 
         FIG. 15  shows the amino acid sequence of the MS2-p65-HSF1 fusion protein of the present invention. A. Amino acid sequence of the MS2-p65-HSF1 (SEQ ID NO: 10) fusion protein including a nuclear localization signal (PKKKRKV, bold (SEQ ID NO: 12)); shaded sequence corresponds to the p65 polypeptide (SEQ ID NO: 8). B. Amino acid sequence of the MS2 peptide (SEQ ID NO: 7). C. Amino acid sequence of the p65 transcription activation domain (SEQ ID NO: 8). D. Amino acid sequence of the HSF1 transcription activation domain (SEQ ID NO: 9); 
         FIG. 16  shows the amino acid sequence of a Cas9 protein lacking nuclease activity (dCas9, SEQ ID NO: 33) used in an embodiment of the present invention. The protein comprises an NLS (PKKKRKV [SEQ ID NO: 12]). This protein comprises the mutations known as D10A (corresponding to position 30 in this sequence) and H840A (corresponding to position 860 in this sequence) (in bold) which inactivate its nuclease activity; 
         FIG. 17  shows the amino acid sequence of the TAG peptide sequence (suntag) which may be fused to a dCas9 protein to enhance transcription of the frataxin gene. A. GCN4 peptide motif (SEQ ID NO: 4) which is repeated 10 times in the TAG peptide sequence. B. Nuclear localization signal (NLS, SEQ ID NO: 12) present in the TAG peptide sequence. C. Complete TAG amino acid sequence (SEQ ID NO: 6) including the NLS followed by 10 GCN4 peptide sequence interspaced with a linker sequence (GSGSG; (SEQ ID NO: 56)); 
         FIG. 18  shows the nucleic acid sequences of exemplary gRNA sequences of the present invention. A. Nucleic acid sequences of gRNAs tested in Examples 1 and 2. DNA sequences encoding the gRNAs were cloned in an expression vector and expressed under the control of the U6 promoter. CrispA to CrispE gRNA sequences correspond to SEQ ID NOs: 57 to 61, respectively. The gRNA guide sequence is underlined (Crisp A to Crisp E gRNA guide sequences correspond to SEQ ID NOs: 69 to 73, respectively). B. Nucleic acid sequences of gRNAs which comprises 2 nucleic acid sequences recognized by the MS2 polypeptide (in bold in MS-2 CrispA, SEQ ID NO: 62). MS2-CrispA to MS2-CrispE gRNA sequences correspond to SEQ ID NOs: 62 to 66. The gRNA guide sequence is underlined (MS2-Crisp A to MS2-Crisp E gRNA guide sequences correspond to SEQ ID NOs: 69 to 73, respectively); 
         FIG. 19  shows the amino acid sequence of the Cas9 nuclease from  Streptococcus pyogenes  (SEQ ID NO: 35; Uniprot Q99ZW2); and 
         FIG. 20  shows a list of mutations in Cas9 and their effect on protein function. Mutation(s) may be selected do design and prepare an inactive Cas9 nuclease which lacks nuclease activity but still binds the target frataxin DNA sequence in the present of a gRNA. 
     
    
    
     DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS 
     Recent discoveries in the field of bacterial immunity have led to the development of a new system for controlling gene expression in cells. Bacterial and archaea have developed adaptive immune defenses termed clustered regularly interspaced short palindromic repeats (CRISPR) systems, which use crRNA and Cas proteins to degrade complementary sequences present in invading viral and plasmid DNA (Mali et al. 2013b). Jinek et al. (Jinek et al. 2012) and Mali et al. (Mali et al. 2013b) have engineered a type II bacterial CRISPR system using custom guide RNA (gRNA) to induce a double strand break in DNA (see  FIG. 1 ). Cong et al. (Gong et al. 2013) and Cho et al. (Cho et al. 2013) have used this system to mutate several genes in mouse and human cells. This CRISPR system has been used to induce mutations in  C. elegans , Zebrafish and in hiPSCs (human induced pluripotent stem cells) and to generated transgenic mice. However the original Cas9 nuclease combined with a gRNA produced frequent off-target mutagenesis (Fu et al. 2013). This problem has been resolved by mutating the Cas9 nuclease into a nickase and using two gRNA to cut both DNA strands (Mali et al. 2013a). The CRISPR system with an inactive Cas9 nuclease coupled or not with the VP64 transcription activation domain has also been used to reduce or enhance gene expression (Perez-Pinera et al. 2013). 
     Applicant shows herein for the first time that the CRISPR system may be used to efficiently increase frataxin protein expression in cells. Various gRNAs targeting the frataxin promoter were shown to increase frataxin expression in cells expressing a mutated (inactive) Cas (e.g., dCas9) nuclease, fused with at least one transcription activation domain (TAD). Frataxin expression was increased not only in normal cells but also in cells from Friedreich Ataxia patients bearing hyper-expansion of a GAA triplet repeat associated with low expression of frataxin. Targeting of the frataxin promoter and increased frataxin expression in these cells was possible without correcting the endogenous defect or modifying the DNA topology and/or its methylation level (e.g., using reagents modifying DNA methylation levels or the like). 
     The present invention relates to inducing or increasing frataxin expression/levels in a cell, and uses thereof. In an aspect, the present invention relates to the design of one or more gRNAs for inducing the expression of frataxin. In a particular aspect, a recombinant fusion protein comprising (a) an inactive Cas nuclease (e.g., dCas9) fused with (b) at least one transcription activation domain (TAD) domain (e.g., VP16, VP64 and VP160), is designed, prepared and introduced into/expressed in a cell together with a gRNA targeting the frataxin promoter, to induce frataxin expression or increase the level of frataxin protein within the cell. In a particular embodiment, the gRNA is modified to include nucleic acid sequences (e.g., two MS2 sequences) to which the MS2 peptide binds and a recombinant fusion protein comprising (a) the MS2 polypeptide fused with (b) at least one transcription activation domain (TAD) domain (e.g., p65 and/or HSF1) are designed and prepared. The fusion proteins are introduced/expressed into a cell together with at least one gRNA targeting the frataxin promoter, to induce frataxin expression or increase the level of frataxin protein within the cell. 
     In another aspect, a recombinant fusion protein comprising (a) an inactive Cas nuclease (e.g., dCas9) polypeptide fused with (b) a polypeptide (TAG) comprising one or more (in an embodiment, at least two) peptide epitopes (e.g., GCN4), is designed and prepared. A further fusion protein which binds to the peptide epitope (antigen binding protein) and which is fused to at least one transcription activation domain (TAD) is also designed and prepared. The fusion proteins are introduced/expressed into a cell together with a gRNA targeting the frataxin promoter, to induce frataxin expression or increase the level of frataxin protein within the cell. 
     The present invention further relates to uses of such induction or increasing frataxin expression/levels in a cell, such as for inducing/increasing expression of the frataxin protein in cells from a subject in need thereof, such as for the treatment of Friedreich ataxia. 
     Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting. 
     Definitions 
     In order to provide clear and consistent understanding of the terms in the instant application, the following definitions are provided. 
     Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described, but methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention. All publications, patent applications, patents, references to sequence database entries, and other references mentioned herein are incorporated by reference in their entirety. 
     As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, un-recited elements or method steps and are used interchangeably with, the phrases “including but not limited to” and “comprising but not limited to”. 
     For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 18-20, the numbers 18, 19 and 20 are explicitly contemplated, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated. 
     “Adeno-associated virus” or “AAV” as used interchangeably herein refers to a small virus belonging to the genus Dependovirus of the Parvoviridae family that infects humans and some other primate species. AAV is not currently known to cause disease and consequently the virus causes a very mild immune response. 
     “Binding region” as used herein refers to the region within a nuclease target region that is recognized and bound by the nuclease. 
     “Coding sequence” or “encoding nucleic acid” as used herein means the nucleic acids (RNA or DNA molecule) that comprise a nucleotide sequence which encodes a protein or gRNA. The coding sequence can further include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered. The coding sequence may be codon optimized. 
     “Complement” or “complementary” as used herein refers to Watson-Crick (e.g., A-T/U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of nucleic acid molecules. “Complementarity” refers to a property shared between two nucleic acid sequences, such that when they are aligned antiparallel to each other, the nucleotide bases at each position will be complementary. 
     “Frataxin” as used herein refers to a protein found in mitochondria (Isoform 1: NCBI NM_000144.4, NP_000135 (210 aa), SEQ ID NO: 1; Isoform 2: NM_181425, NP_852090 (196 aa), SEQ ID NO: 2; Isoform 3: NM_001161706, NP_001155178, (171 aa), SEQ ID NO: 3; Uniprot Q16595; ENTREZ 2395; Ensembl ENSG00000165060; OMIM: 606829). The Frataxin gene or “FRX gene” (NCBI reference number NG_008845; NCBI Gene ID 2395) as used interchangeably herein is at locus 9q21.1. 
     “Fusion protein” as used herein refers to a chimeric protein created through the joining of two or more nucleic acid/genes that each originally coded for separate proteins or polypeptides. The translation of the fusion nucleic acid/gene results in a single polypeptide with functional properties derived from each of the original proteins. 
     A “TAG” in the context of the present invention is a polypeptide sequence comprising at least one peptide epitope (in embodiments at least 3, 4, 5, 6, 7, 10, 20, 24, or more peptide epitopes) which bind to a protein having antigen binding activity (e.g., an antibody or single chain variable fragment). 
     “Mutant gene” or “mutated gene” as used interchangeably herein refers to a gene (e.g., frataxin gene) that has undergone a detectable mutation. A mutant frataxin gene in the context of FRDA comprises an abnormal amount of GAA trinucleotide repeats (about 150 to more than 1700 repeats) in the first intron of the frataxin gene. The presence of these repeats affects the normal transmission and expression of the gene, thereby leading to reduced expression of functional frataxin protein. 
     “Normal frataxin gene” as used herein refers to a frataxin gene (e.g., NCBI reference number; NG_008845; NCBI Gene ID 2395, SEQ ID NO: 87) that has not undergone a change, such as a loss, gain, or exchange of genetic material. The normal gene undergoes normal gene transmission and gene expression. A “normal” frataxin gene in the context of the present invention is a frataxin gene which encodes a wild-type, functional frataxin protein and which expresses a level of frataxin protein which substantially corresponds to the level of frataxin protein normally found in healthy subjects (e.g., subjects not suffering from FRADA). Healthy subjects comprise less than 35 GAA repeats in intron 1 of the frataxin gene and the average amount of repeats in healthy subjects is between about 6-34 repeats. 
     “Promoter” as used herein means a synthetic or naturally-derived molecule which is capable of conferring, activating or enhancing expression of a nucleic acid in a cell. A promoter may comprise one or more specific transcriptional regulatory sequences to further enhance expression and/or to alter the spatial expression and/or temporal expression of same. A promoter may also comprise distal enhancer or repressor elements, which may be located as much as several thousand base pairs from the start site of transcription. A promoter may be derived from sources including viral, bacterial, fungal, plants, insects, and animals. A promoter may regulate the expression of a gene component constitutively, or differentially with respect to cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents. Representative examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40 late promoter and the CMV IE promoter. The “frataxin promoter” enables the expression of the frataxin protein in cells (see for example  FIG. 2  (SEQ ID NO: 11) for a partial sequence of the human frataxin promoter and the frataxin gene sequence (SEQ ID NO: 87; NG_00845) for the complete promoter sequence). In an embodiment, the U6 promoter is used to express one or more gRNAs in a cell. 
     “Subject” and “patient” as used herein interchangeably refers to any vertebrate, including, but not limited to, a mammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate (for example, a monkey, such as a cynomolgous or rhesus monkey, chimpanzee, etc.) and a human). In some embodiments, the subject may be a human or a non-human. In an embodiment, the subject or patient may suffer from FRDA and has a mutated frataxin gene having an abnormal hyper-expansion of trinucleotide repeats in intron 1 of the frataxin gene. The subject or patient may be undergoing other forms of treatment. 
     “Target gene” as used herein refers to any nucleotide sequence encoding a known or putative gene product. The target gene may be a mutated gene involved in a genetic disease. 
     “Vector” as used herein means a nucleic acid sequence containing an origin of replication. A vector may be a viral vector, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome. A vector may be a DNA or RNA vector. A vector may be a self-replicating extrachromosomal vector, and preferably, is a DNA plasmid. For example, the vector may comprise nucleic acid sequence(s) that/which encode(s) a fusion protein of the present invention such as a dCas9-TAD (e.g., dCas9-VP64 or dCas9-VP160) a dCas9-polyGCN4 (suntag), a MS2-TAD (e.g., MS2-p65-HFS1), or an antigen-binding protein-TAD (e.g., scFV-VP64 or scFV-VP160) fusion protein or that encodes at least one gRNA (e.g. SEQ ID NOs: 57-66). Alternatively, the vector may comprise nucleic acid sequence(s) that/which encode(s) one or more of the above fusion protein and at least one gRNA nucleotide sequence of the present invention (e.g., a gRNA A, C and/or D or a gRNA comprising SEQ ID NOs:57-66). A vector for expressing one or more gRNA will comprise a “DNA” sequence of the gRNA. 
     Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those that are well known and commonly used in the art. The meaning and scope of the terms should be clear; in the event however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. 
     CRISPR System 
     “Clustered Regularly Interspaced Short Palindromic Repeats” and “CRISPRs”, as used interchangeably herein refers to loci containing multiple short direct repeats that are found in the genomes of approximately 40% of sequenced bacteria and 90% of sequenced archaea. The CRISPR system is a microbial nuclease system involved in defense against invading phages and plasmids that provides a form of acquired immunity. This defensive pathway has three steps. First a copy of the invading nucleic acid is integrated into the CRISPR locus. Short segments of foreign DNA, called spacers, are incorporated into the genome between CRISPR repeats, and serve as a ‘memory’ of past exposures. Next, CRISPR RNAs (i.e., crRNAs, the endogenous bacterial RNA that confers target specificity) are transcribed from this CRISPR locus. The crRNAs are then incorporated into effector complexes, where the crRNA guides the complex to the invading nucleic acid and the Cas proteins degrade this nucleic acid. There are several pathways of CRISPR activation, one of which requires a tracrRNA which plays a role in the maturation of crRNA. TracrRNA is complementary to and base pairs with a pre-crRNA forming an RNA duplex. This is cleaved by RNase III, an RNA-specific ribonuclease, to form a crRNA/tracrRNA hybrid (gRNA). This hybrid acts as a guide for the endonuclease Cas9, which cleaves the invading nucleic acid. 
     Cas9 forms a complex with the 3′ end of the gRNA, and the protein-RNA pair recognizes its genomic target by complementary base pairing between the 5′ end of the gRNA sequence and a predefined 20 bp DNA sequence, known as the protospacer. This complex is directed to homologous loci of pathogen DNA via regions encoded within the crRNA, i.e., the protospacers, and protospacer-adjacent motifs (PAMs) within the pathogen genome. The non-coding CRISPR array is transcribed and cleaved within direct repeats into short crRNAs containing individual spacer sequences, which direct Cas nucleases to the target site (protospacer). By simply exchanging the 20 bp recognition sequence of the expressed gRNA, the Cas9 nuclease can be directed to new genomic targets. CRISPR spacers are used to recognize and silence exogenous genetic elements in a manner analogous to RNAi in eukaryotic organisms. 
     Three classes of CRISPR systems (Types I, II and III effector systems) are known. The Type II effector system carries out targeted DNA double-strand break in four sequential steps, using a single effector enzyme, Cas9, to cleave dsDNA. Compared to the Type I and Type III effector systems, which require multiple distinct effectors acting as a complex, the Type II effector system may function in alternative contexts such as eukaryotic cells. The Type II effector system consists of a long pre-crRNA, which is transcribed from the spacer-containing CRISPR locus, the Cas9 protein, and a tracrRNA, which is involved in pre-crRNA processing. The tracrRNAs hybridize to the repeat regions separating the spacers of the pre-crRNA, thus initiating dsRNA cleavage by endogenous RNase III. This cleavage is followed by a second cleavage event within each spacer by Cas9, producing mature crRNAs that remain associated with the tracrRNA and Cas9, forming a Cas9:crRNA-tracrRNA complex. 
     The Cas9:crRNA-tracrRNA complex unwinds the DNA duplex and searches for sequences matching the crRNA to cleave. Target recognition occurs upon detection of complementarity between a “protospacer” sequence in the target DNA and the remaining spacer sequence in the crRNA. Cas9 mediates cleavage of target DNA if a correct protospacer-adjacent motif (PAM) is also present at the 3 ′ end of the protospacer. For protospacer targeting, the sequence must be immediately followed by the protospacer-adjacent motif (PAM), a short sequence recognized by the Cas9 nuclease that is required for DNA cleavage. Members of the Cas9 family require different protospacer adjacent motifs (PAM) (Hou et al. 2013). As noted above, the PAM is the sequence of nucleotides which must follow the nucleotide sequence targeted by the gRNA. Different Type II systems have differing PAM requirements. Accordingly, selection of a specific polynucleotide target sequence (e.g., on the frataxin promoter) by a gRNA will be based on the recombinant dCas protein used. 
     The  S. pyogenes  CRISPR system may have the PAM sequence for this Cas9 (SpCas9) as 5’-NRG-3′, where R is either A or G, and characterized the specificity of this system in human cells. A unique capability of the CRISPR/Cas9 system is the straightforward ability to simultaneously target multiple distinct genomic loci by co-expressing a single Cas9 protein with two or more sgRNAs (at least one, two, three, four, five, six seven, eight, nine or ten gRNAs). For example, the  Streptococcus pyogenes  Type II system naturally prefers to use an “NGG” sequence, where “N” can be any nucleotide, but also accepts other PAM sequences, such as “NAG” in engineered systems. Similarly, the Cas9 derived from  Neisseria meningitidis  (NmCas9) normally has a native PAM of NNNNGATT, but has activity across a variety of PAMs, including a highly degenerate NNNNGNNN PAM. 
     CRISPR/Cas9-Based Systems 
     An engineered form of the Type II effector system of  Streptococcus pyogenes  was shown to function in human cells for genome engineering. In this system, the Cas9 protein was directed to genomic target sites by a synthetically reconstituted “guide RNA” (“gRNA”, also used interchangeably herein as a chimeric single guide RNA (“sgRNA”)), which is a crRNA-tracrRNA fusion that obviates the need for RNase III and crRNA processing in general. It comprises a “gRNA guide sequence” or “gRNA target sequence” and a Cas9 recognition sequence, which is necessary for Cas (e.g., Cas9) binding to the targeted gene. The gRNA guide sequence is the sequence which confers specificity. It hybridizes with (i.e., it is complementary to) the opposite strand of a target sequence (i.e., it corresponds to the RNA sequence of a DNA target sequence). 
     Provided herein are CRISPR/Cas9-based engineered systems for use in increasing frataxin expression in cells. The CRISPR/Cas9-based engineered systems of the present invention are designed to target the frataxin promoter to increase frataxin gene transcription and ultimately to increase the level of frataxin protein in cells, such as cells from subjects suffering from FRDA or having an hyper trinucleotide expansion in the first intron of the frataxin gene (which reduces frataxin expression compared to subjects not having the hyper-expansion in intro 1). The CRISPR/Cas (e.g. CRISPR/Cas9)-based systems of the present invention include an inactive Cas (e.g. Cas9) fusion protein (lacking nuclease activity, dCas (e.g., dCas9)) and at least one gRNA. 
     In an embodiment, the inactive Cas9 (dCas9) fusion protein comprises an inactive Cas 9 (dCas9) polypeptide domain lacking nuclease activity and a polypeptide domain that has a different activity that which is endogenous to Cas9. 
     In a first aspect, the polypeptide domain that has a different activity that which is endogenous to Cas9 has transcription activation activity (the dCas9 polypeptide domain is fused to a transcriptional activator). In an embodiment, the dCas9 fusion protein comprises a polypeptide domain comprising one or more (at least one) transcriptional activation domains (TAD) (dCas9/TAD). The fact that the dCas9/TAD has transcription activation activity allows increasing frataxin expression when used in combination with a gRNA targeting the frataxin promoter sequence. 
     In a second aspect, the CRSPR/dCas9 system of the present invention is a 3-component modified CRISPR system. It comprises a gRNA; a dCas9 fusion protein and a fusion protein having antigen-binding activity (e.g., an antibody or a scFv fragment), which comprises transcription activation domain (e.g., VP16, VP64, VP160). In this system, the dCas9 fusion protein comprises an inactive Cas 9 (dCas9) polypeptide domain lacking nuclease activity and a polypeptide domain comprising at least one epitope. Preferably, multiple repetitions of a peptide epitope (e.g., GCN4 peptide, SEQ ID NO: 4) are fused to dCas9. This peptide epitope is recognized by a fusion protein having antigen-binding activity. The presence of multiple repetitions of the peptide epitopes on the dCas9 fusion protein allows to recruit multiple transcription activation domains (TADs) at a single target site on the frataxin promoter, thereby further increasing its expression. (Tanenbaum et al., Cell, 2014 and  FIG. 13 ); 
     In a third aspect, the CRISPR/Cas9-based system of the present invention is also a 3-component system. It may comprise a modified gRNA (MS2-gRNA), a dCas9-TAD fusion protein and a MS2-TAD fusion protein. The MS2/TAD fusion protein comprises an MS2 polypeptide (SEQ ID NO: 7) and at least one transcription activation domain. The MS2-gRNA comprises at least one MS2 nucleotide sequence (SEQ ID NO: 68,  FIG. 18 ). In an embodiment, two MS2 nucleotide sequences are included in the gRNA: one on the tetraloop and one on the stem loop of the gRNA. In an embodiment, a fusion protein comprising the MS2 peptide and two TADs (e.g., the p65 and HSF1 transcription activation domains (TADs)) binds to each of the MS2 nucleotide sequences included in the gRNA. The presence of multiple TADs (e.g., the VP64/VP160 on the dCas9/TAD protein and the p65 and HSF1 on the MS2/TAD fusion protein) produces a strong synergistic effect on the transcription of the gene (frataxin) targeted by the gRNA. (Konermann et al., Nature, 2014; see  FIG. 14 ). 
     In accordance with the present invention, the target frataxin gene may or may not have a hyper-expansion of GAA triplets in intron 1. In an embodiment, the target frataxin gene is a mutated gene comprising at least 50, at least 75, at least 100, at least 150, at least 200, at least 250, at least 300, at least 500 or more GAA triplets in intron 1. In an embodiment, the target frataxin gene is from a subject suffering from FRDA. In an embodiment, the target gene is a normal gene from a healthy subject not suffering from FRDA. 
     a. Inactive Cas9 (dCas) 
     The wild type Cas9 protein is an endonuclease that cleaves nucleic acid and is encoded by the CRISPR loci and is involved in the Type II CRISPR system. Cas9 proteins are produced by numerous species of bacteria including  Streptococcus pyogene  (Deltcheva et al. 2011),  Streptococcus thermophiles  (Sapranauskas et al. 2011) and  Neisseria meningitides  (Hou et al. 2013). The CRISPR/Cas9-based system of the present invention uses an inactive Cas9 protein (dCas9) or a dCas9 fusion protein with no endonuclease activity. As used herein, “iCas9” and “dCas9” both refer to a Cas9 protein that has its nuclease activity inactivated. The inactive Cas9 protein used in accordance with the present invention may be derived from a Cas9 from any bacterial or archaea species. The dCas9 fusion protein (i.e., dCas9 polypeptide domain in the dCas9 fusion protein) of the present invention is thus i) derived from any naturally occurring Cas, and ii) lacks a nuclease activity normally associated with such protein. The dCas9 fusion protein comprises at least one amino acid mutation which knocks out nuclease activity of Cas9 ( FIG. 19 , SEQ ID NO: 35). 
     In an embodiment, the amino acid sequence of the dCas9 fusion protein of the present invention comprises an amino acid sequence at least 95% (in embodiments at least 96%, 97%, 98% or 99%) identical to the Cas9 sequence set forth in  FIG. 19  (SEQ ID NO: 35) and comprises at least one amino acid mutation which knocks out nuclease activity of Cas9. 
     Several mutations are known to cause inactivation of Cas9 nuclease activity (see for example  FIG. 20 ). The dCas9 fusion protein of the present invention may comprise any mutation or combination of mutations (such as those listed in  FIG. 20 ) as long as it lacks nuclease activity but still provides acceptable target DNA binding activity. In embodiments, the at least one amino acid mutation may be at amino acid position 10 and/or 840, for example the at least one amino acid mutation may be at least one of D10A and H840A with respect to the amino acid numbering of the Cas9 sequence shown in  FIG. 19 . In other embodiments, the at least one mutation is selected from those listed in  FIG. 20 . In an embodiment, the at least one mutation is at amino acid position 10, 15, 66, 70, 74, 78, 840 or any combination thereof with respect to the amino acid numbering of the Cas9 amino acid sequence set forth in  FIG. 19  (SEQ ID NO: 35). In an embodiment, the amino acid sequence of the dCas9 protein of the present invention comprises an amino acid sequence at least 95% (in embodiments at least 96%, 97%, 98% or 99%) identical to the dCas9 sequence set forth in  FIG. 16  (SEQ ID NO: 33). In another embodiment, the amino acid sequence of the dCas9 protein of the present invention comprises an amino acid sequence at least 95% identical to amino acids 10 to 1407 of the sequence set forth in  FIG. 3  (SEQ ID NO: 32) 
     In a particular embodiment, the dCas9 polypeptide domain in the dCas9 fusion protein is derived from  Streptococcus pyogenes  Cas9 (SEQ ID NO: 35) and comprises mutations at amino acid positions 10 and/or 840 (e.g., the mutations D10A and/or H840A). An example of a dCas9 protein is shown in  FIG. 16  (SEQ ID NO: 33)). 
     dCas9 Fusion Proteins and MS2 Fusion Proteins 
     The CRISPR/Cas9-based system/Frataxin targeting system of the present invention includes one or more (at least one) fusion proteins. The fusion protein may comprise two heterologous polypeptide domains, wherein the first polypeptide domain comprises an inactive Cas9 polypeptide lacking nuclease activity (dCas9) and the second polypeptide domain has an activity heterologous to the dCas9 polypeptide, such as transcription activation activity, or comprises at least one peptide epitope interacting with a second fusion protein having antigen binding activity (antigen-binding protein). 
     In a first aspect, the fusion protein is a dCas9/TAD fusion protein which comprises a dCas9 polypeptide domain, as described above, fused to a second polypeptide domain that has transcription activation activity, such as a transcription activation domain (TAD), to induce the transcription of the frataxin gene and expression of the frataxin protein when in presence of an appropriate gRNA targeting the frataxin promoter. Thus, in an embodiment, the dCas9/TAD fusion protein of the present invention comprises one or more (i.e., at least one) of a “transcription activation domain” or “trans-activating domain” (TAD), which contains binding sites for other proteins (e.g., transcription co-regulators) and functions for activating transcription of the target frataxin gene and expression of the frataxin protein. 
     For example, gene expression of endogenous mammalian genes, such as human frataxin gene, may be achieved by targeting a dCas9/TAD fusion protein to the frataxin promoter via one or more gRNAs. The transactivation domain may include a VP16 protein, multiple VP16 proteins, such as a VP48 domain, VP64 domain, VP160 domain or the like. For example, the fusion protein may be dCas9-VP64 or dCas9-VP160 (e.g., SEQ ID NO: 32 or SEQ ID NO: 33) or a functional variant thereof enabling the transcription of the frataxin promoter when in the presence of at least one suitable gRNA. 
     In another aspect the fusion protein is a dCas9/TAG fusion protein comprising an inactive Cas9 (dCas9) polypeptide domain and a TAG polypeptide domain, comprising at least one polypeptide epitope, e.g., GCN4 polypeptide (SEQ ID NO: 4)) recognized by a second fusion protein or polypeptide having antigen binding activity and transcription activation activity. In an embodiment, the dCas/TAG fusion protein comprises 10 peptide epitopes. In an embodiment, the TAG polypeptide domain of the dCas9 fusion protein comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least, 8, at least 9, at least 10, at least 12, at least 16, at least 20 or at least 24 peptide epitopes. In an embodiment, the dCas9/TAD fusion protein comprises 10 GCN4 epitopes. In an embodiment, the TAG polypeptide domain of the dCas9/TAG fusion protein comprises the sequence as set forth in SEQ ID NO: 5 or 6). 
     In an embodiment, the second fusion protein (antigen binding fusion protein) has transcription activation activity and is able to bind to the dCas9/TAG fusion protein comprising the at least one peptide epitope. In an embodiment, the antigen-binding fusion protein is an antibody or antigen-binding protein or polypeptide coupled to one or more (at least one) TAD ( FIG. 13 ). In an embodiment, the antigen-binding protein is a single chain variable fragment (ScFv), which is coupled with VP64 or VP160 (Tanenbaum et al., 2014). The one or more transactivation domains allow recruitment of transcriptional activators and effectors at the target site on the frataxin promoter to increase frataxin expression. 
     In an embodiment, the dCas9/TAG comprises a dCas9 polypeptide fused to multiple repetitions of the peptide epitope (e.g., GCN4 peptide epitope EELLSKNYHLENEVARLKK, SEQ ID NO: 4, see  FIG. 17 ), interspaced with a linker (e.g., GSGSG, SEQ ID NO: 56). 
     In a third aspect, the present invention provides a MS2/TAD-fusion protein to be used in conjunction with a modified gRNA and a dCas9/TAD fusion protein having transcription activating activity (comprising one or more TADS). The MS2/TAD fusion protein comprises an MS2 domain (e.g., SEQ ID NO: 7, which recognizes specific nucleotide sequences on a modified gRNA) and at least two TADs (see for example  FIG. 15 ). In an embodiment, the MS2/TAD fusion protein comprises the MS2 polypeptide, the p65 polypeptide (SEQ ID NO: 8,  FIG. 15C ) and HSF1 polypeptide (SEQ ID NO: 9,  FIG. 15D ). In an embodiment, the MS2 fusion protein comprises the sequence set forth in  FIG. 15A  (SEQ ID NO: 10), or a variant thereof which has transcription activation activity and which binds to a modified gRNA comprising the MS2 nucleotide sequence ( FIG. 18  and SEQ ID NO: 68). Preferably, the MS2 fusion protein comprises a linker between the MS2 domain and the TADs.  FIG. 15  shows the amino acid sequence of the MS2-p65-HSF1 fusion protein according to an embodiment of the present invention. 
     gRNAs 
     The gRNA provides the targeting specificity of the present invention. The gRNA provides both targeting specificity and scaffolding/binding ability for of the CRISPR/dCas9-based system. The gRNA of the present invention does not exist in nature, i.e., is a non naturally-occurring nucleic acid. 
     The gRNA of the present invention generally comprises (or consists of) a “gRNA guide sequence” or “gRNA target sequence” and a Cas (e.g., Cas9) recognition sequence, which is necessary for Cas/dCas (e.g., Cas9/dCas9) binding to the targeted frataxin gene. In a particular embodiment, the Cas9 recognition sequence comprises (or consists of) the sequence (SEQ ID NO: 67). The gRNA of the present invention may comprise any variant of this sequence, provided that it allows for the binding of the dCas9 fusion protein of the present invention on the frataxin target promoter sequence. 
     A “gRNA target sequence”, or “gRNA guide sequence” is the nucleic acid sequence that binds to the target frataxin gene. This sequence immediately precedes (i.e., is 5′ to) the PAM sequence in the genomic frataxin DNA. It is what gets put into a gRNA expression construct (e.g., vector/plasmid/AVV), it does NOT include the PAM sequence. The “PAM” (Protospacer Adjacent Motif) is the nucleic acid sequence, that immediately follows (is contiguous) to the target sequence on the frataxin gene but is not in the gRNA. 
     A “target region”, “target sequence” or “protospacer” as used interchangeably herein refers to the region of the target gene which is targeted by the CRISPR/dCas9-based system, without the PAM. The CRISPR/dCas9-based system may include at least one gRNA, wherein the gRNAs target different DNA sequences on the frataxin promoter. The target DNA sequences may be overlapping. The target sequence or protospacer is followed by (e.g., (is contiguous to) a PAM sequence at the 3′ end of the protospacer. In an embodiment, the target sequence is immediately adjacent to the PAM sequence and is located on the 5′ end of the PAM. 
     The gRNA comprises a “gRNA guide sequence” or “gRNA target sequence”, which corresponds to the target sequence on the frataxin DNA promoter that is followed by a PAM sequence. The gRNA may comprise a “G” at the 5′ end of the polynucleotide sequence. The CRISPR/dCas9 system of the present invention may use gRNA of varying lengths. The gRNA may comprise at least a 10 nts, at least 11 nts, at least a 12 nts, at least a 13 nts, at least a 14 nts, at least a 15 nts, at least a 16 nts, at least a 17 nts, at least a 18 nts, at least a 19 nts, at least a 20 nts, at least a 21 nts, at least a 22 nts, at least a 23 nts, at least a 24 nts, at least a 25 nts, at least a 30 nts, or at least a 35 nts of the target frataxin promoter DNA sequence which is followed by a PAM sequence. The “gRNA guide sequence” or “gRNA target sequence” may be least 17 nucleotides (17, 18, 19, 20, 21, 22, 23), preferably between 17 and 30 nts long, more preferably between 18-22 nucleotides long. In an embodiment, gRNA guide sequence is between 10-40, 10-30, 12-30, 15-30, 18-30, or 10-22 nucleotides long. The PAM sequence may be “NGG”, where “N” can be any nucleotide. gRNA may target any region of the frataxin promoter, which is immediately upstream (contiguous, adjoining, in 5′) to a PAM (e.g., NGG) sequence including an enhancer region of the frataxin gene (Ensembl ENSG00000165060; OMIM: 606829, see also partial frataxin promoter sequence on  FIG. 2 ). In an embodiment, the gRNA may target any region, which is followed by a PAM identified on the frataxin promoter sequence of  FIG. 2  (SEQ ID NO: 11)). 
     Although a perfect match between the “gRNA guide sequence” and the DNA strand to which it binds on the frataxin promoter is preferred, a mismatch between a gRNA guide sequence and a target base on the frataxin promoter sequence is also permitted as along as it still allows for an increase in frataxin expression. A “seed” sequence of between 8-12 consecutive nucleotides on the gRNA perfectly complementary to the target gene sequence is preferred for proper recognition of the target sequence on the frataxin promoter. The remainder of the guide sequence may comprise one or more mismatch. 
     In general, gRNA activity is inversely correlated with the number of mismatches. Preferably, the gRNA of the present invention comprises 7 mismatches, 6 mismatches, 5 mismatches, 4 mismatches, 3 mismatches, more preferably 2 mismatches, or less, and even more preferably no mismatch, with the corresponding target frataxin promoter sequence. The smaller the number of nucleotides in the gRNA the smaller the number of mismatches tolerated. The binding affinity is thought to depend on the sum of matching gRNA-DNA combinations. 
     Non-limiting examples of gRNAs guide sequences are presented in  FIG. 18 . In a particular embodiment, the “gRNA guide sequence” comprises or consists of a nucleic acid sequence as set forth in SEQ ID NOs: 69-73. In an embodiment, the gRNA comprises the target sequence of nucleotides positions 4246-4264; positions 4652-4670; Positions 4841-4859; positions 5005-5023; or positions 5089-5107; of the frataxin promoter polynucleotide sequence having NCBI reference number NG_008845 (see also  FIG. 2  and SEQ ID NO: 87). 
     In an embodiment, the gRNA target sequence on the frataxin promoter ends at position 5107 (or comprises a sequence including position 5107 and upstream) with respect to the nucleotide sequence shown in  FIG. 2  (i.e., is adjacent to the NGG sequence beginning at position 5108). In an embodiment, the gRNA target sequence on the frataxin promoter ends at position 4859 (or comprises a sequence including position 4859 and upstream) with respect to the nucleotide sequence shown in  FIG. 2  (i.e., is adjacent to the NGG sequence beginning at position 4860). In an embodiment, the gRNA target sequence on the frataxin promoter ends at position 4670 (or comprises a sequence including position 4670 and upstream) with respect to the nucleotide sequence shown in  FIG. 2  (i.e., is adjacent to the NGG sequence beginning at position 4671). In an embodiment, the gRNA target sequence on the frataxin promoter ends at position 4701 (or comprises a sequence including position 4701 and upstream) with respect to the nucleotide sequence shown in  FIG. 2  (i.e., is adjacent to the NGG sequence beginning at position 4702. In an embodiment, the gRNA target sequence on the frataxin promoter ends at position 44742 (or comprises a sequence including position 4742 and upstream) with respect to the nucleotide sequence shown in  FIG. 2  (i.e., is adjacent to the NGG sequence beginning at position 4743). 
     In an embodiment, the gRNA target sequence on the frataxin promoter is not rich in polyG or polyC. In an embodiment, the gRNA target sequence on the frataxin promoter does not comprise more than one PAM (e.g., NGG sequence). In an embodiment, the gRNA target sequence on the frataxin promoter does not include an NGG (although it is adjacent to a PAM). In an embodiment, the gRNA target sequence comprises between 10-22 consecutive nucleotides, preferably 18-22 consecutive nucleotides upstream of and adjacent to a PAM (NGG sequence) located between: 
     nucleotides 3961-4258; 
     nucleotides 4268-49513; 
     nucleotides 4268-4996 
     nucleotides 5027-5150; 
     nucleotides 4268-4595; 
     nucleotides 4621-4996; 
     nucleotides 4561-4996; or 
     nucleotides 4280-5000; 
     of the frataxin promoter sequence shown in  FIG. 2  (NG_008845; SEQ ID NO: 87). 
     The number of gRNAs administered to or expressed in a cell or subject in accordance with the methods of the present invention may be at least 1 gRNA, at least 2 gRNAs, at least 3 gRNAs at least 4 gRNAs, at least 5 gRNAs, at least 6 gRNAs, at least 7 gRNAs, at least 8 gRNAs, at least 9 gRNAs, at least 10 gRNAs, at least 11 gRNAs, at least 12 gRNAs, at least 13 gRNAs, at least 14 gRNAs, at least 15 gRNAs, at least 16 gRNAs, at least 17 gRNAs, or at least 18 gRNAs. The number of gRNAs administered to or expressed in a cell may be between at least 1 gRNA and at least 15 gRNAs, at least 1 gRNA to and least 10 gRNAs, at least 1 gRNA and at least 8 gRNAs, at least 1 gRNA and at least 6 gRNAs, at least 1 gRNA and at least 4 gRNAs, at least 1 gRNA to and least 3 gRNAs, at least 2 gRNA and at least 5 gRNAs, at least 2 gRNA and at least 3 gRNAs. 
     In a particular embodiment a combination of at least two gRNAs comprising a gRNA guide sequence as shown in  FIG. 18  (SEQ ID NOs: 69-73) are used together to increase frataxin expression. In a particular embodiment, a combination of at least two gRNAs comprising a gRNA guide sequence comprising (or consisting of) the sequence i) AGCUGGGAAGUUCUUCCUG (CRISP A, SEQ ID NO: 69); ii) ACACAAGGCAUCCGUCUCC (CRISP C, SEQ ID NO: 71); or iii) UAUUUAUUGUGCACUUAAC (CRISP D, SEQ ID NO: 72) are used together to increase frataxin expression in a cell. 
     Trans-Activating Domains (TADs) 
     “Trans-activating domain(s)” or “transcription activation domain(s)” (TAD(s)) used in the context of the present invention refers to a polypeptide which has transcriptional activity (is able to activate or enhance transcription of a target nucleic acid). TADs are typically named after their amino acid composition. These amino acids are either essential for the activity or simply the most abundant in the TAD. Transactivation by the Gal4 transcription factor is mediated by acidic amino acids, whereas hydrophobic residues in Gcn4 play a similar role. Hence, the TADs in Gal4 and Gcn4 are referred to as acidic or hydrophobic activation domains, respectively. 
     Nine-amino-acid transactivation domain (9 aaTAD) defines a novel domain common to a large superfamily of eukaryotic transcription factors represented by Gal4, Oaf1, Leu3, Rtg3, Pho4, Gln3, Gcn4 in yeast and by p53, NFAT, NF-κB and VP16 in mammals. Prediction for 9 aa TADs (for both acidic and hydrophilic transactivation domains) is available online from ExPASy™ and EMBnet™ databases. 
     KIX domain of general coactivators Med15(Gal11) interacts with 9 aa TAD transcription factors Gal4, Pdr1, Oaf1, Gcn4, VP16, Pho4, Msn2, Ino2 and P201. 9aaTAD is a common transactivation domain which recruits multiple general coactivators (e.g., TAF9, MED15, CBP/p300 and GCN5). Accordingly, non-limiting examples of TAD that may be used in accordance with the present invention include TAD from Gal4, Pdr1, Oaf1, Gcn4, Pho4, Msn2, Ino2, P201, p53, Gli3, Pdr3, CREB, Rtg3, VP16, MLL, E2A, HSF1, NF-IL6, NFAT1 and NF-κβ. Other non-limiting examples of TAD include TAD from the SRF, TFAP2 or SP1 transcription factor, for which target sequences have been identified in the frataxin promoter (Li et al., 2010). Of course, the choice of a TAD will depend on numerous factors including the specific type of cells in which the gene will be expressed as well as the nature of the gene. Furthermore, one can appreciate that more than one TAD may be included in a fusion protein of the present invention (e.g., dCas9/TAD construct; MS2/TAD peptide construct or antigen-binding protein/TAD construct) of the present invention. Similarly, Also, a plurality of TADs having the same or different peptide sequences can be used in accordance with the present invention. In an embodiment, the TAD fused to the dCas9 protein/MS2 fusion or antibody/fragment thereof of the present invention is VP160 which corresponds to 10 times the sequence of the VP16 TAD (see  FIGS. 4 and 5 ). In an embodiment, the TAD has the sequence DALDDFDLDML (SEQ ID NO: 17) repeated 1-15 times, preferably 5 to 12 times, more preferably 10 times. Each TAD repeat is normally insterspaced by small linker sequences (e.g., two amino acid linker GS). 
                     TABLE 1                  Examples of TADs from transcription factors.                         Transcription               factor   9aaTAD   Peptide-KIX interaction (NMR data)               P53 TAD1   E TFSD LWKL (SEQ ID NO: 19)   LSPEETFSD LWKLPE (SEQ ID NO: 102)               P53 TAD2   D DIEQ WFTE (SEQ ID NO: 20)   QAMDDLMLSPD DIEQ WFTEDPGPD (SEQ ID               NO: 103)               MLL   S DIMD FVLK (SEQ ID NO: 21)   DCGNILPS DIMD FVLKNTP (SEQ ID NO: 104)               EA2   D LLDF SMMF (SEQ ID NO: 22)   PVGTDKELSDLLDF SMMFPLPVT (SEQ ID NO:               105)               Rtg3   E TLDF SLVT (SEQ ID NO: 23)   E2A Homolog               CREB   RKILNDLSS (SEQ ID NO: 24)   RREILSRRPSYRKILNDLSSDAP (SEQ ID NO:               106)               CREBαB6   E AILAELKK (SEQ ID NO: 25)   CREB-mutant binding to KIX               Gli3   DDVVQYLNS (SEQ ID NO: 26)   TAD homology to CREB/KIX               Gal4   DDVYNYLFD (SEQ ID NO: 27)   Pdr1 and Oaf1 homolog               Oaf1   DLFDYDFLV (SEQ ID NO: 28)   DLFDYDFLV (SEQ ID NO: 107)               Pip2   DFFDYDLLF (SEQ ID NO: 29)   Oaf1 homolog               Pdr1   EDLYSILWS (SEQ ID NO: 30)   EDLYSILWSDWY (SEQ ID NO: 108)               Pdr3   TDLYHTLWN (SEQ ID NO: 31)   Pdr1 homolog                    
Nuclear Localization Signal
 
     The fusion recombinant proteins of the present invention may also comprises at least one Nuclear Localization Signal (NLS). Accordingly, as used herein the expression “nuclear localization signal” or “NLS” refers to an amino acid sequence, which ‘directs’ a protein for import into the cell nucleus by nuclear transport. Typically, this signal consists of one or more short sequences of positively charged lysines or arginines exposed on the protein surface. Different nuclear localized proteins may share the same NLS. An NLS has the opposite function of a nuclear export signal, which targets proteins out of the nucleus. Classical NLSs can be further classified as either monopartite or bipartite. The first NLS to be discovered was the sequence PKKKRKV (SEQ ID NO: 12) in the SV40 Large T-antigen (a monopartite NLS). The NLS of nucleoplasmin, KR[PAATKKAGQA]KKKK (SEQ ID NO: 13), is the prototype of the ubiquitous bipartite signal: two clusters of basic amino acids, separated by a spacer of about 10 amino acids. 
     There are many other types of NLS, which are the to be “non-classical”, such as the acidic M9 domain of hnRNP A1, the sequence KIPIK in yeast transcription repressor Mata2, the complex signals of U snRNPs as well as a recently identified class of NLSs known as PY-NLSs. Thus, any type of NLS (classical or non-classical) may be used in accordance with the present invention as long as it targets the protein of interest into the nucleus of a target cell. Preferably, the NLS is derived from the simian virus 40 large T antigen. In an embodiment, the NLS of the dCas9 fusion proteins of the present invention comprises the following amino acid sequence: SPKKKRKVEAS (SEQ ID NO: 14). In an embodiment the NLS comprises the sequence KKKRKV (SEQ ID NO: 15). In an embodiment, the NLS comprises the sequence SPKKKRKVEASPKKKRKV (SEQ ID NO: 16). In another embodiment, the NLS comprises the sequence KKKRK (SEQ ID NO: 109). 
     Protein Transduction Domains 
     The fusion recombinant proteins of the present invention (e.g., dCas9/TAD, dCas9/TAG or MS2/TAD) of the present invention may also be coupled to a protein transduction domain to ensure entry of the protein into the target cells. Alternatively the gene coding for the gRNA and for the fusion proteins of the present invention may be delivered to the cells using various vectors, e.g., viral vectors. 
     Protein transduction domains (PTD) may be of various origins and allow intracellular delivery of a given therapeutic by facilitating the translocation of the protein/polypeptide into a cell membrane, organelle membrane, or vesicle membrane. PTD refers to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates traversing a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. A PTD attached to another molecule facilitates the molecule traversing a membrane, for example going from extracellular space to intracellular space, or cytosol to within an organelle including the mitochondria. In an embodiment, a PTD is covalently linked to the amino terminus of a recombinant protein of the present invention. In another embodiment, a PTD is covalently linked to the carboxyl terminus of a recombinant protein of the present invention. Exemplary protein transduction domains include but are not limited to a minimal undecapeptide protein transduction domain (corresponding to residues 47-57 of HIV-1 TAT comprising YGRKKRRQRRR (SEQ ID NO: 37); a polyarginine sequence comprising a number of arginines sufficient to direct entry into a cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines); a VP22 domain (Zender et al. 2002); an  Drosophila  Antennapedia protein transduction domain (Noguchi et al. 2003); a truncated human calcitonin peptide (Trehin et al. 2004); RRQRRTSKLMKR (SEQ ID NO: 38); Transportan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 39); KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO: 40); and RQIKIWFQNRRMKWKK (SEQ ID NO: 41). Further exemplary PTDs include but are not limited to, KKRRQRRR (SEQ ID NO: 42), RKKRRQRRR (SEQ ID NO: 43); or an arginine homopolymer of from 3 arginine residues to 50 arginine residues. 
     Other non-limiting examples of PTD include an endosomal escape peptide. Non-limiting examples of such endosomal escape peptides are listed in the Table 2 below. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Endosomal escape peptides 
               
            
           
           
               
               
               
               
            
               
                 Peptide 
                 Primary sequence 
                 Mechanism 
                 References 
               
               
                   
               
               
                 DT 
                 VGSSLSCINLDWDVIRDKTKTKIE 
                 Fusion 
                 (Kakimoto et al. 2009) 
               
               
                   
                 SLKEHGPIKNKMSESPNKTVSEE 
                   
                 (SEQ ID NO: 44) 
               
               
                   
                 KAKQYLEEFHQTALEHPELSELKT 
                   
                   
               
               
                   
                 VTGTNPVFAGANYAAWAVNVAQ 
                   
                   
               
               
                   
                 VIDSETADNLEKTTAALSILPGIGS 
                   
                   
               
               
                   
                 VMGIADGAVHHNTEEIVAQSIALS 
                   
                   
               
               
                   
                 SLMVAQAIPLVGELVDIGFAAYNF 
                   
                   
               
               
                   
                 VESIINLFQVVHNSYNRPAYSPG 
                   
                   
               
               
                   
               
               
                 GALA 
                 WEAALAEALAEALAEHLAEALAE 
                 Membrane 
                 (Kakudo et al. 2004) 
               
               
                   
                 ALEALAA 
                 destabilization, pore 
                 (SEQ ID NO: 45) 
               
               
                   
                   
                 formation and flip- 
                   
               
               
                   
                   
                 flop of membrane 
                   
               
               
                   
                   
                 lipids 
                   
               
               
                   
               
               
                 PEA 
                 VLAGNPAKHDLDIKPTVISHRLHF 
                 Pore formation 
                 (Fominaya, Uherek and Wels 
               
               
                   
                 PEGGSLAALTAHQACHLPLETFT 
                   
                 1998) (SEQ ID NO: 46) 
               
               
                   
                 RHRQPRGWEQLEQCGYPVQRLV 
                   
                   
               
               
                   
                 ALYLAARLSWNQVDQVIRNALAS 
                   
                   
               
               
                   
                 PGSGGDLGEAIREQPEQARLALT 
                   
                   
               
               
                   
               
               
                 INF-7 
                 GLFEAIEGFIENGWEGMIDGWYG 
                 Membrane fusion 
                 (El-Sayed, Futaki and 
               
               
                   
                 C 
                 and destabilization 
                 Harashima 2009) 
               
               
                   
                   
                   
                 (SEQ ID NO: 47) 
               
               
                   
               
               
                 LAH4 
                 KKALLALALHHLAHLALHLALALK 
                 Membrane 
                 (Kichler et al. 2003) 
               
               
                   
                 KA 
                 destabilization 
                 (SEQ ID NO: 48) 
               
               
                   
               
               
                 CM18 
                 KWKLFKKIGAVLKVLTTG 
                 Membrane 
                 (Salomone et al. 2012) 
               
               
                   
                   
                 destabilization 
                 (SEQ ID NO: 49) 
               
               
                   
               
               
                 HGP 
                 LLGRRGWEVLKYWWNLLQYWS 
                 Pore formation and 
                 (Kwon, Bergen and Pun 2008)  
               
               
                   
                 QEL 
                 fusion 
                 (SEQ ID NO: 50) 
               
               
                   
               
               
                 H5WYG 
                 GLFHAIAHFIHGGWH 
                 Membrane 
                 (Midoux et al. 1998) 
               
               
                   
                 GLIHGWYG 
                 destabilization 
                 (SEQ ID NO: 51) 
               
               
                   
               
               
                 HA2 
                 GLFGAIAGFIENGWEGMIDGWYG 
                 Membrane fusion 
                 (Lorieau, Louis and Bax 2010) 
               
               
                   
                   
                 and destabilization 
                 (SEQ ID NO: 52) 
               
               
                   
               
               
                 EB1 
                 LIRLWSHLIHIWFQNRRLKWKKK 
                 Membrane 
                 (Lundberg et al. 2007) 
               
               
                   
                   
                 destabilization 
                 (SEQ ID NO: 53) 
               
               
                   
               
            
           
         
       
     
     In an embodiment, the protein transduction domain is TAT or Pep-1. In an embodiment, the protein transduction domain is TAT and comprises the sequence SGYGRKKRRQRRRC (SEQ ID NO: 36). In another embodiment, the protein transduction domain is TAT and comprises the sequence YGRKKRRQRRR (SEQ ID NO: 37). In another embodiment, the protein transduction domain is TAT and comprises the sequence KKRRQRRR (SEQ ID NO: 42). In another embodiment, the protein transduction domain is Pep-1 and comprises the sequence KETWWETWWTEWSQPKKKRKV (SEQ ID NO: 54). In addition or alternatively to the above-mentioned protein transduction domains, the fusion recombinant protein of the present invention may be coupled to liposomes to further facilitate translocation into the cell and mitochondria. 
     Genetic constructs encoding a dCas/TAD protein in accordance with the present invention can be made using either conventional gene synthesis or modular assembly. A humanized Cas9 nucleic acid is available at the public, not-for-profit repository by AddGene (for example AddGene plasmid pAC154). 
     In an aspect, the gRNAs; dCas/TAD; dCas/TAG and/or MS2/TAD recombinant fusion proteins of the present invention may be used to increase/induce expression of the frataxin nucleic acid and the frataxin protein in cells. As used herein, the expression “increasing” in “increasing the expression of frataxin in a cell” is meant to include circumstances where, in the absence of a gRNA and of a Cas9 recombinant protein of the present invention, the frataxin protein is not expressed at all in the cell and where the cell already expresses a certain amount (baseline amount) of frataxin protein. It comprises increasing/enhancing expression of frataxin in cells expressing no frataxin, a normal level of frataxin or an abnormal/lower level of frataxin (as compared to normal conditions). 
     In an embodiment, the gRNA and recombinant fusion proteins of the present invention may be used to increase transcription of the frataxin promoter and expression of the frataxin protein in cells from a subject in need thereof. 
     As used herein, “a subject in need thereof” is a subject, which may benefit from an increased expression of the frataxin protein or of increased levels of the frataxin protein. Non-limiting examples of a subject in need thereof include a subject having cells showing a reduced level of frataxin expression or activity as compared to cells from a normal subject. In an embodiment, the subject in need thereof is a subject having an abnormal number of trinucleotide repeats in intron 1 of the frataxin gene. In an embodiment, the number of trinucleotide repeats is 35 or more, 65 or more, 75 or more, 85 or more, 100 or more, 110 or more, 125 or more, 150 or more, 175 or more, 200 or more, 225 or more, 250 or more, 300 or more, 350 or more, 500 or more or 1000 or more. In an embodiment, the subject in need thereof suffers from Friedreich ataxia. In an embodiment, the subject is a mammal, preferably, a human. 
     In an embodiment, the present invention relates to a method of increasing frataxin expression in a subject in need thereof comprising administering to the subject an effective amount of at least one gRNA and at least one (in embodiments at least two) recombinant fusion protein of the present invention (e.g., dCas/TAD; dCas/TAG; antigen-binding protein/TAD and/or MS2/TAD). In an embodiment, the recombinant protein is specifically formulated for crossing the plasma membrane and reaching the nucleus. In an embodiment, the present invention provides a composition comprising at least one (in embodiments at least two) recombinant fusion protein (e.g., (e.g., dCas/TAD; dCas/TAG; antigen-binding protein/TAD and/or MS2/TAD) of the present invention together with a pharmaceutically or physiologically acceptable carrier. 
     In an embodiment, the present invention relates to a method of increasing frataxin levels in a subject in need thereof, comprising administering to the subject an effective amount of at least one of the fusion proteins of the present invention ((e.g., dCas/TAD; dCas/TAG; antigen-binding protein/TAD and/or MS2/TAD) together with at least one gRNA targeting the frataxin promoter. In an embodiment, the present invention provides a composition comprising a recombinant fusion protein of the present invention together with a pharmaceutically or physiologically acceptable carrier. 
     Optimization of Codon Degeneracy 
     Because Cas nuclease proteins are normally expressed in bacteria, it may be advantageous to modify their nucleic acid sequences for optimal expression in eukaryotic cells (e.g., mammalian cells) when designing and preparing Cas recombinant proteins. This has already been done for the embodiment of the dCas9/TAD protein of the present invention described herein. 
     Accordingly, the following codon chart (Table 3) may be used, in a site-directed mutagenic scheme, to produce nucleic acids encoding the same or slightly different amino acid sequences of a given nucleic acid: 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Codons encoding the same amino acid 
               
            
           
           
               
               
               
               
            
               
                 Amino Acids 
                   
                   
                 Codons 
               
               
                   
               
               
                 Alanine 
                 Ala 
                 A 
                 GCA GCC GCG GCU 
               
               
                   
               
               
                 Cysteine 
                 Cys 
                 C 
                 UGC UGU 
               
               
                   
               
               
                 Aspartic acid 
                 Asp 
                 D 
                 GAC GAU 
               
               
                   
               
               
                 Glutamic acid 
                 Glu 
                 E 
                 GAA GAG 
               
               
                   
               
               
                 Phenylalanine 
                 Phe 
                 F 
                 UUC UUU 
               
               
                   
               
               
                 Glycine 
                 Gly 
                 G 
                 GGA GGC GGG GGU 
               
               
                   
               
               
                 Histidine 
                 His 
                 H 
                 CAC CAU 
               
               
                   
               
               
                 Isoleucine 
                 Ile 
                 I 
                 AUA AUC AUU 
               
               
                   
               
               
                 Lysine 
                 Lys 
                 K 
                 AAA AAG 
               
               
                   
               
               
                 Leucine 
                 Leu 
                 L 
                 UUA UUG CUA CUC CUG CUU 
               
               
                   
               
               
                 Methionine 
                 Met 
                 M 
                 AUG 
               
               
                   
               
               
                 Asparagine 
                 Asn 
                 N 
                 AAC AAU 
               
               
                   
               
               
                 Proline 
                 Pro 
                 P 
                 CCA CCC CCG CCU 
               
               
                   
               
               
                 Glutamine 
                 Gln 
                 Q 
                 CAA CAG 
               
               
                   
               
               
                 Arginine 
                 Arg 
                 R 
                 AGA AGG CGA CGC CGG CGU 
               
               
                   
               
               
                 Serine 
                 Ser 
                 S 
                 AGC AGU UCA UCC UCG UCU 
               
               
                   
               
               
                 Threonine 
                 Thr 
                 T 
                 ACA ACC ACG ACU 
               
               
                   
               
               
                 Valine 
                 Val 
                 V 
                 GUA GUC GUG GUU 
               
               
                   
               
               
                 Tryptophan 
                 Trp 
                 W 
                 UGG 
               
               
                   
               
               
                 Tyrosine 
                 Tyr 
                 Y 
                 UAC UAU 
               
               
                   
               
            
           
         
       
     
     Sequence Similarity 
     “Homology” and “homologous” refers to sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing each position in the aligned sequences. A degree of homology between nucleic acid or between amino acid sequences is a function of the number of identical or matching nucleotides or amino acids at positions shared by the sequences. As the term is used herein, a nucleic acid sequence is “substantially homologous” to another sequence if the two sequences are substantially identical and the functional activity of the sequences is conserved (as used herein, the term “homologous” does not infer evolutionary relatedness, but rather refers to substantial sequence identity, and thus is interchangeable with the terms “identity”/“identical”). Two nucleic acid sequences are considered substantially identical if, when optimally aligned (with gaps permitted), they share at least about 50% sequence similarity or identity, or if the sequences share defined functional motifs. In alternative embodiments, sequence similarity in optimally aligned substantially identical sequences may be at least 60%, 70%, 75%, 80%, 85%, 90% or 95%. For the sake of brevity, the units (e.g., 66, 67 . . . 81, 82, . . . 91, 92% . . . ) have not systematically been recited but are considered, nevertheless, within the scope of the present invention. 
     Substantially complementary nucleic acids are nucleic acids in which the complement of one molecule is substantially identical to the other molecule. Two nucleic acid or protein sequences are considered substantially identical if, when optimally aligned, they share at least about 70% sequence identity. In alternative embodiments, sequence identity may for example be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 98% or at least 99%. Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, such as the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, the search for similarity method of Pearson and Lipman (Pearson and Lipman 1988), and the computerized implementations of these algorithms (such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wis., U.S.A.). Sequence identity may also be determined using the BLAST algorithm, described in Altschul et al. (Altschul et al. 1990) 1990 (using the published default settings). Software for performing BLAST analysis may be available through the National Center for Biotechnology Information (through the internet at http://www.ncbi.nlm.nih.gov/). The BLAST algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. Initial neighborhood word hits act as seeds for initiating searches to find longer HSPs. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction is halted when the following parameters are met: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. One measure of the statistical similarity between two sequences using the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. In alternative embodiments of the invention, nucleotide or amino acid sequences are considered substantially identical if the smallest sum probability in a comparison of the test sequences is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001. 
     An alternative indication that two nucleic acid sequences are substantially complementary is that the two sequences hybridize to each other under moderately stringent, or preferably stringent, conditions. Hybridization to filter-bound sequences under moderately stringent conditions may, for example, be performed in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.2×SSC/0.1% SDS at 42° C. (Ausubel 2010). Alternatively, hybridization to filter-bound sequences under stringent conditions may, for example, be performed in 0.5 M NaHPO4, 7% SDS, 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C. (Ausubel 2010). Hybridization conditions may be modified in accordance with known methods depending on the sequence of interest (Tijssen 1993). Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point for the specific sequence at a defined ionic strength and pH. 
     In another aspect, the invention further provides a nucleic acid encoding the above-mentioned fusion proteins ((e.g., dCas/TAD; dCas/TAG; antigen-binding protein/TAD and/or MS2/TAD) and gRNAs. In an embodiment, the nucleic acid encoding dCas9 is as set forth in SEQ ID NO: 93. In an embodiment, the nucleic acid encoding for VP16 is as set forth in SEQ ID NO: 94. In an embodiment, the nucleic acid encoding VP160 is as set forth in SEQ ID NO: 95. In an embodiment, the nucleic acid encoding a GCN4 epitope with the linker is as set forth in SEQ ID NO: 96. In an embodiment, the nucleic acid encoding a GCN4 epitope epitope is as set forth in SEQ ID NO: 97. In an embodiment, the nucleic acid encoding a MS2 polypeptide is as set forth in SEQ ID NO: 98. In an embodiment, the nucleic acid encoding a p65 TAD is as set forth in SEQ ID NO: 99. In an embodiment, the nucleic acid encoding a HSF1 polypeptide is as set forth in SEQ ID NO: 100. The invention also provides a vector comprising the above-mentioned nucleic acid. In an embodiment, the vector further comprises a transcriptional regulatory element operably-linked to the above-mentioned nucleic acid. A first nucleic acid sequence is “operably-linked” with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably-linked to a coding sequence if the promoter affects the transcription or expression of the coding sequences. Generally, “operably-linked” DNA sequences are contiguous and, where necessary to join two protein coding regions, in reading frame. However, since, for example, enhancers generally function when separated from the promoters by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably-linked but not contiguous. “Transcriptional regulatory element” is a generic term that refers to DNA sequences, such as initiation and termination signals, enhancers, and promoters, splicing signals, polyadenylation signals, which induce or control transcription of protein coding sequences with which they are operably-linked. 
     As indicated above, gRNAs and fusion proteins of the present invention (e.g., dCas/TAD, dCas9/TAG, MS2/TAD and antigen binding protein/TAD) of the present invention may be delivered into cells using one or more various viral vectors. Accordingly, preferably, the above-mentioned vector is a viral vector for introducing the gRNA and/or fusion protein of the present invention in a target cell. Non-limiting examples of viral vectors include retrovirus, lentivirus, Herpes virus, adenovirus or Adeno Associated Virus, as well known in the art. 
     The modified MV vector preferably targets one or more cell types expressing frataxin and affected in FRDA subjects. Accordingly, the modified AAV vector may have enhanced cardiac (e.g., cardiomyocytes), skeletal muscle, neuronal, liver, and/or pancreatic (e.g., Langerhans cells) tissue tropism. The modified AAV vector may be capable of delivering and expressing the at least one gRNA and fusion proteins of the present invention in the cell of a mammal. For example, the modified AAV vector may be an AAV-SASTG vector (Piacentino et al. (2012) Human Gene Therapy 23:635-646). The modified AAV vector may preferably deliver gRNAs and fusion proteins to neuronal, skeletal, pancreatic (e.g., Langherans cells) and cardiac (e.g., cardiomyocytes) muscle in vivo. The modified AAV vector may be based on one or more of several capsid types, including AAVI, AAV2, AAV5, AAV6, AAV8, and AAV9. The modified AAV vector may be based on AAV2 pseudotype with alternative muscle-tropic AAV capsids, such as AAV2/1, AAV2/6, AAV2/7, AAV2/8, AAV2/9, AAV2.5 and AAV/SASTG vectors that efficiently transduce skeletal muscle or cardiac muscle by systemic and local delivery. In an embodiment, the modified AAV vector is a AAV-DJ. In an embodiment, the modified AAV vector is a AAV-DJ8 vector. In an embodiment, the modified AAV vector is a AAV2-DJ8 vector. 
     In yet another aspect, the present invention provides a cell (e.g., a host cell) comprising the above-mentioned nucleic acid and/or vector. The invention further provides a recombinant expression system, vectors and host cells, such as those described above, for the expression/production of a recombinant protein, using for example culture media, production, isolation and purification methods well known in the art. 
     In another aspect, the present invention provides a composition (e.g., a pharmaceutical composition) comprising the above-mentioned gRNA and dCas9/TAD recombinant protein. In an embodiment, the composition further comprises one or more pharmaceutically acceptable carriers, excipients, and/or diluents. 
     As used herein, “pharmaceutically acceptable” (or “biologically acceptable”) refers to materials characterized by the absence of (or limited) toxic or adverse biological effects in vivo. It refers to those compounds, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the biological fluids and/or tissues and/or organs of a subject (e.g., human, animal) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. 
     The present invention further provides a kit or package comprising at least one container means having disposed therein at least one of the above-mentioned gRNAs, fusion proteins; vectors, cells, frataxin targeting systems, combinations or compositions, together with instructions for increasing frataxin expression or levels in a cell or for treatment of Friedreich ataxia in a subject. 
     The present invention is illustrated in further details by the following non-limiting examples. 
     EXAMPLE 1 
     gRNA Efficiently Promotes Frataxin Expression in 293T Cells 
     Expression plasmids containing a gene coding for one of the gRNA under the U6 promoter and the dCas9 nuclease under the CAG promoter have been produced (see table 4 below and  FIGS. 4 and 18 ). The original plasmid was obtained from Addgene (pAC154-dual-dCas9VP160-sgExpression-plasmid no. 48240). The resulting dCas9 protein comprises two NLS, one HA tag and a VP160 transactivation domain (See  FIG. 3 , (SEQ ID NO: 32)). The gRNAs produced target different nucleotide sequences in the promoter of the human frataxin gene (ACCESSION NG_008845, see Table 4 below and  FIGS. 2 and 18 ). 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 gRNAs targeting the human frataxin promoter 
               
            
           
           
               
               
               
               
            
               
                   
                 Position of targeted nucleo- 
                 Targeted nucleotide  
                 SEQ ID 
               
               
                 gRNA 
                 tides with reference to the 
                 sequence in the 
                 NO: 
               
               
                 name 
                 sequence shown in FIG. 2 
                 frataxin promoter 
                 (RNA/DNA) 
               
               
                   
               
               
                 A 
                 5089-5107 
                 AGCTGGGAAGTTCTTCCTG 
                 69/88 
               
               
                   
               
               
                 B 
                 5005-5023 
                 TCCCTTGGGTCAGGGGTCC 
                 70/89 
               
               
                   
               
               
                 C 
                 4841-4859 
                 ACACAAGGCATCCGTCTCC 
                 71/90 
               
               
                   
               
               
                 D 
                 4652-4670 
                 TATTTATTGTGCACTTAAC 
                 72/91 
               
               
                   
               
               
                 E 
                 4246-4264 
                 GCTACTTGGAAGGCTGAAA 
                 73/92 
               
               
                   
               
            
           
         
       
     
     The efficient targeting of the promoter of the human frataxin gene by these gRNAs was initially tested in 293T cells. These cells were transfected with a plasmid coding for i) an active Cas9 nuclease from  Streptococcus pyogenes  and ii) a gRNA. When a gRNA binds correctly to its target sequence in the promoter, it forms a complex with the Cas9 nuclease. This results in a double strand break (DSB) in the promoter ( FIG. 1 ). This DSB is spontaneously repaired by Non-Homologous End Joining (NHEJ), an imperfect repair process that results in micro-insertion or micro-deletions (INDELs) of some base pairs. The presence or absence such INDELs was detected by amplifying by PCR (oligos: aatctatcaacaatagaggcaaggca (forward, SEQ ID NO: 74) and cagctcccagcgtagctaca (reverse, SEQ ID NO: 75) part of the frataxin promoter. The amplicons were heated at 95° C. to separate the 2 DNA strands and slowly cooled at RT to favor the formation of heterodimers if INDELs were present in some of the amplicons. These amplicons were then exposed to the Surveyor enzyme that cuts double strands DNA, which have mismatches resulting in additional bands visualized on agarose gel stained with RedSafe™. The Surveyor enzyme results are illustrated in  FIG. 6 . These results indicate that gRNA A, C and D were able to bind effectively with their targeted sequence on the frataxin promoter. 
     The induction of frataxin gene expression was initially tested by transfecting 293T cells with one or several plasmids pAC154 each coding for a different gRNAs and for the same HA-dCas9-VP160, an inactive Cas9 nuclease (dCas9) coupled with 10 VP16 sequences that act as transcription activation domains (see for Example  FIG. 4  for pAC154 sequence coding for gRNA A and VP160 TAD and  FIG. 8  for a schematic representation of the complex). The 293T cells were seeded into 6 plates the day before transfection at densities of 3.5×10 5  cells/well. 72 hours after transfection, total proteins were extracted from cells. The expression of the HA-dCas9-VP160 was initially confirmed by Western blot using an anti-HA antibody ( FIG. 7 ). 
     If the gRNAs and the HA-dCas9-VP160 proteins are able to attach to the frataxin promoter, this complex should drive the expression of the frataxin gene. The increased expression of the frataxin gene was first investigated by qRT-PCR amplification of the frataxin mRNA.  FIG. 9  illustrates that gRNAs increased the expression of frataxin at the mRNA level (results normalized with the 18S RNA). The results were also normalized relative to cells transfected with a plasmid coding for eGFP rather than for the HA-dCas9-VP160 protein. Primers used for amplification are shown in Table 5 below. 
     The gRNAs and the HA-dCas9-VP160 proteins were also able to increase the expression of the frataxin protein ( FIG. 10 ) in the 293T cells transfected with one or several plasmids pAC154. Frataxin protein expression was normalized using β-actin as an internal standard. The mAb used to detect frataxin was #18A5DB1 from Mitosciences. 
     EXAMPLE 2 
     gRNAs and dCas9-VP160 Significantly Increase the Expression of the Human Frataxin Gene in Fibroblasts of Friedreich Patients 
     The ability of gRNAs and dCas9-VP160 to increase frataxin expression was next evaluated in cells of Friedreich Ataxia patients which normally express low levels of frataxin. Various combinations of gRNAs A, B, C, D and E also increased by 30 to 40% the frataxin mRNA in fibroblasts from a patient suffering from Friedreich ataxia Indeed, nucleofection of plasmids coding for dCas9-VP160 and for gRNA A, B and C increased the frataxin mRNA compared with a control nucleofected with a plasmid coding for eGFP ( FIG. 11 ). The frataxin mRNA was amplified by PCR (using primers defined in Table 5 and normalized with 18S rRNA or GAPDH). The Friedreich fibroblasts used for this experiment were obtained from Coriell Institute for Medical Research (GM 04078) and have 541 and 420 trinucleotide repeats in intron 1 of each allele of the gene respectively. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Primers used 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                 T 
                   
                   
               
               
                   
                   
                   
                   
                 anneal- 
                 Primer sequences 
                   
               
               
                 Gene 
                   
                   
                 Size 
                 ing 
                 5′→3′ 
                 SEQ ID 
               
               
                 Symbol 
                 Description 
                 GenBank 
                 (bp) 
                 (° C.) 
                 Forward/Reverse 
                 NOs 
               
               
                   
               
               
                 Hs FXN 
                 
                   Homo sapiens 
                 
                 NM_000144 
                 106 
                 57 
                 AAGCCATACACGTTTGAGGACT 
                 SEQ ID 
               
               
                   
                 frataxin (FXN), 
                   
                   
                   
                 A/ 
                 NO: 76 
               
               
                   
                 region targeted 
                   
                   
                   
                 TTGGCGTCTGCTTGTTGATCA 
                 SEQ ID 
               
               
                   
                 common to 
                   
                   
                   
                   
                 NO: 77 
               
               
                   
                 isoforms 1, 2 
                   
                   
                   
                   
                   
               
               
                   
                 and 3 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 Hs 
                 
                   Homo sapiens 
                 
                 NM_002046 
                 194 
                 57 
                 GGCTCTCCAGAACATCATCCCT/ 
                 SEQ ID 
               
               
                 GAPDH 
                 glyceraldehyde- 
                   
                   
                   
                 ACGCCTGCTTCACCACCTTCTT 
                 NO: 78/ 
               
               
                   
                 3-phosphate 
                   
                   
                   
                   
                 SEQ ID 
               
               
                   
                 dehydrogenase 
                   
                   
                   
                   
                 NO: 101 
               
               
                   
               
               
                 Hs 18S 
                 Homo sapiens 
                 NR_003286 
                 119 
                 57 
                 ACGGACCAGAGCGAAAGCATT/ 
                 SEQ ID 
               
               
                   
                 18S ribosomal 
                   
                   
                   
                 TCCGTCAATTCCTTTAAGTTTCA 
                 NO: 79/ 
               
               
                   
                 RNA (Rn18s) 
                   
                   
                   
                 GCT 
                 SEQ ID 
               
               
                   
                   
                   
                   
                   
                   
                 NO: 80 
               
               
                   
               
               
                 Mm Hprt1 
                 
                   Mus musculus 
                 
                 NM_013556 
                 106 
                 57 
                 CAGGACTGAAAGACTTGCTCGA 
                 SEQ ID 
               
               
                   
                 hypoxanthine 
                   
                   
                   
                 GAT/ 
                 NO: 81/ 
               
               
                   
                 guanine 
                   
                   
                   
                 CAGCAGGTCAGCAAAGAACTTA 
                 SEQ ID 
               
               
                   
                 phosphoribosyl 
                   
                   
                   
                 TAGC 
                 NO: 82 
               
               
                   
                 transferase 1 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 Mm 
                 
                   Mus musculus 
                 
                 NM_008084 
                 123 
                 57 
                 ACGGGAAGCTCACTGGCATGG/ 
                 SEQ ID 
               
               
                 GAPDH 
                 glyceraldehyde- 
                   
                   
                   
                 ATGCCTGCTTCACCACCTTCTT 
                 NO: 83/ 
               
               
                   
                 3-phosphate 
                   
                   
                   
                 G 
                 SEQ ID 
               
               
                   
                 dehydrogenase 
                   
                   
                   
                   
                 NO: 84 
               
               
                   
               
               
                 Mm 18S 
                 18S ribosomal 
                 NR_003278 
                 119 
                 57 
                 TGGATACCGCAGCTAGGAATAA 
                 SEQ ID 
               
               
                   
                 RNA (Rn18s) 
                   
                   
                   
                 TG/ 
                 NO: 85/ 
               
               
                   
                   
                   
                   
                   
                 TCACCTCTAGCGGCGCAATAC 
                 SEQ ID 
               
               
                   
                   
                   
                   
                   
                   
                 NO: 86 
               
               
                   
               
            
           
         
       
     
     Results obtained at the mRNA level were also confirmed at the protein level. The combination of gRNAs A, B and C also increased by almost 20% the frataxin protein in fibroblasts from the same Friedreich patient ( FIG. 12 ). Co-nucleofection of pAC154 plasmid coding for gRNAs A, B and C, or co-nucleofection of B, C and D increased frataxin protein expression compared to control cells nucleofected with a plasmid coding for GFP from Amaxa inc. Frataxin protein expression was normalized using β-actin as an internal standard. The mAb used to detect frataxin was #18A5DB1 from Mitosciences. 
     Such an increase of frataxin protein in the subject cells may be used to reduce or prevent the symptoms associated with Friedreich ataxia. 
     EXAMPLE 3 
     Production and In Vivo Testing of AAV Vectors Coding for at Least One gRNA and Fusion Proteins dCAs9/TAD; dCas9/TAG, MS2-TAD and Antigen-Binding Protein/TAD 
     AAV constructs such as AAV-DJ (or AAV-DJ8) for the gRNAs (alone or in combination) and fusion proteins to increase frataxin expression may be prepared. These AAV are produced at the Molecular Tools Platform of the Centre de Recherche Institut Universitaire en Santé Mentale de Québec. The plasmid to produce the DJ serotype AAV form is available from Cell Biolabs inc. One or multiple AAV vectors can be used to deliver the gRNAFrat and the TAD. For example, the dCas9-VP64, dCas9-VP160 or dCas9-SunTag may be separated in two parts sharing an overlapping sequence. This permits a recombination of the 2 parts following infection of a cell. Several articles describing these dual AAVs have been published by Duan and colleagues (Bostwick et al., Gosh et al., 2007, Gosh et al., 2008 and Gosh et al., 2011). Further AAV-DJ containing one or several gRNAFrat (or gRNAFrat-MS2) under the U6 and/or other promoter(s) can also be prepared. The AAV vectors may be delivered via the superficial temporal vein at different doses (1 to 10 E10 vector genomes per mouse for initial testing) to groups of ten 5 days old YG8sR mice (Pook et al., (2001); Al-Mandawi et al., (2004) and Al-Mandawi et al., (2006) and Virmouni et al., (2015); e.g., stock #024097). Control mice are injected with a AAV vector (e.g., AAV-DJ) coding for an irrelevant gRNA. Since the dCas9-VP64, dCas9-VP160 or dCas9-SunTag may be immunogenic, the YG8sR mouse is immunosuppressed with Tacrolimus for this experiment. The mice are sacrificed 1 month later. The RNA and the proteins are extracted from the brain, spinal cord, spinal ganglions, heart, liver and muscles for quantification of the frataxin mRNA and protein. This method allows to confirm induction of frataxin expression in vivo. 
     EXAMPLE 4 
     Specificity of Gene Expression Induced by the CRISPR System 
     The RNA-Seq technique permits to investigate the expression of the whole human or mouse genome (Wang et al., Pepke et al., Sultan et al., and Citrulli et al.). Briefly, this technique converts the mRNA into cDNA. These cDNAs are then fragmented and oligonucleotides are attached to allow sequencing of 100 bp of each DNA fragment with the Illumina sequencer. For each sample, about 50 million 100 bp sequences should be obtained. Thus RNA-Seq provides a precise quantitative evaluation of the expression of all genes. Even the mRNA expressed only once per cell and alternative splicings are identified by this method. 
     Three groups may be used for this experiment: 1) normal mice, 2) YG8sR mice and 3) YG8sR mice (Pook et al., (2001); Al-Mandawi et al., (2004) and Al-Mandawi et al., (2006) and Virmouni et al., (2015)) treated with the AAV vectors. The RNA-Seq technique is used to compare the expression of all genes in specific tissue such as the brain, spinal ganglia and the heart. This allows to verify whether frataxin expression is restored and to what level of the normal control. This technique also permits to detect whether the treatment induced any abnormal gene expression in the various tissues, which could be due to off-target effects and potential toxicity of the CRISPR system. This will thus permit to assess the specificity of the treatment and identify the metabolic consequence of restoring the frataxin expression. 
     EXAMPLE 5 
     Investigating the Possible Immune Response Against dCas9-VP64, dCas9-VP160, dCas9-SunTag or MS2-p65-HSF1 
     Fusions proteins of the present invention (e.g., dCas9-VP64, dCas9-VP160, dCas9-SunTag and MS2-p65-HSF1) are foreign protein for the mice (and human subjects). The newborn mice may not develop an immune response because their immune system is immature. However, this is not the case of adult mice and of FRDA patients. To investigate the potential immune response against these proteins, the AAV vectors can be administered i.v. to 6 months old YG8sR mice (Pook et al., (2001); Al-Mandawi et al., (2004) and Al-Mandawi et al., (2006) and Virmouni et al., (2015)). One month after the systemic delivery of an AAV derived vector (AAV-DJ), the presence of a humoral and cellular immune response against the proteins coded by the viral vector is investigated. 
     To detect the humoral response, the serum is obtained from the mice one month after the CRISPR treatment. The presence of antibodies in those sera, against the proteins extracted from the heart of mice treated or not with the CRISPR, is determined by for example western blot using as the primary antibody the mouse serum and a second anti-mouse IgG coupled to HRP. 
     To detect a potential cellular immune response, a muscle biopsy is obtained from mice treated or not with the CRISPR. Myoblasts, which are good antigen presenting cells, are grown from the satellite cells present in these muscle biopsies. These myoblasts are infected in vitro with the same AAV used in the CRISPR treatment. The presence of a cellular immune response against the myoblasts expressing one of the foreign proteins is determined by using a IFNγ ELISPOT assay with the T lymphocytes derived from the spleen of mice that received or not the CRISPR treatment. 
     EXAMPLE 6 
     Long-Term In Vivo Study in the YG8sR Mouse Model of FRDA 
     The CRISPR/dCas9 systems based treatments of the present invention can also be investigated in the YG8sR mice (Pook et al., (2001); Al-Mandawi et al., 2004 and Al-Mandawi et al., 2006) for a longer time period (i.e., 1 year). For these experiments, ten YG8sR mice are injected at 5 days of age with the AAV vectors of the CRISPR/dCas9 systems of the present invention. Ten control YG8sR mice receive a saline injection. The expression of frataxin is quantified in the white blood cells every month. At every 2 months during the one-year follow-up, the mouse activity is investigated. The heart function of treated mice (stroke volume, cardiac output, systolic and diastolic diameters) is investigated echography, as done in (Gerard et al., 2014). 
     Following the mouse sacrifice, the weight of the heart is measured to heart hypertrophy and treatment success. Absence of iron accumulation in the heart and brain is also investigated. Mitochondrial enzyme activity, aconitase activity, oxidative stress etc. in heart and brain using methods previously described may also be determined. The organs of mice of each group is sent to Jackson laboratory for examination to detect any potential toxicity from the treatment. 
     Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the invention as defined in the appended claims. 
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         Al-Mandawi, S. et al. GAA repeat instability in Friedreich ataxia YAC transgenic mice. Genomics 84, 301-310 (2004). 
         Al-Mandawi, S. et al. GAA repeat expansion mutation mouse models of Friedreich ataxia exhibit oxidative stress leading to progressive neuronal and cardiac pathology.  Genomics  88, 580-590 (2006). 
         Altschul, S. F., W. Gish, W. Miller, E. W. Myers &amp; D. J. Lipman (1990) Basic local alignment search tool.  J Mol Biol,  215, 403-10. 
         Annoni, A., Goudy, K., Akbarpour, M., Naldini, L. &amp; Roncarolo, M. G. Immune responses in liver-directed lentiviral gene therapy. Translational research: the journal of laboratory and clinical medicine 161, 230-240, doi:10.1016/j.trs1.2012.12.018 (2013). 
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