NZ710497B2 - Methods and pharmaceutical composition for the treatment and the prevention of cardiomyopathy due to energy failure - Google Patents

Abstract

The present invention relates to a method for preventing or treating cardiomyopathy due to energy failure in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a vector which comprises a nucleic acid sequence of a gene that can restore energy failure. More particularly, the invention relates to a method for preventing or treating a cardiomyopathy associated with Friedreich ataxia in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a vector which comprises a frataxin (FXN) encoding nucleic acid. In a particular embodiment, the vector is an AAVrh10 vector and is formulated to be administered at a dose of about 5x10^12 vg/kg. ilure. More particularly, the invention relates to a method for preventing or treating a cardiomyopathy associated with Friedreich ataxia in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a vector which comprises a frataxin (FXN) encoding nucleic acid. In a particular embodiment, the vector is an AAVrh10 vector and is formulated to be administered at a dose of about 5x10^12 vg/kg.

Description

S AND PHARMACEUTICAL COMPOSITION FOR THE TREATMENT AND THE PREVENTION OF CARDIOMYOPATHY DUE TO ENERGY FAILURE FIELD OF THE INVENTION: The present invention relates a method for preventing or treating cardiomyopathy due to energy failure in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a vector which comprises a nucleic acid sequence of a gene that can restore energy failure.

More particularly, the invention relates to a method for preventing or treating a cardiomyopathy associated with eich ataxia in a subject in need thereof, comprising stering to said subject a therapeutically effective amount of a vector which ses a frataxin (FXN) encoding nucleic acid.

BACKGROUND OF THE INVENTION: Friedreich ataxia (FRDA), an autosomal progressive recessive neurodegenerative disorder ated with cardiomyopathy, is caused by reduced sion of the mitochondrial protein, in [V. ano et al., 1996 and V. Campuzano et al., 1997].

The mopathy associated with FRDA is hypertrophic. As the disease progresses, there is a natural tion from hypertrophy to dilation, with death of cardiomyocytes replaced by fibrotic tissue g to systolic and diastolic dysfianction [R. M. Payne et al., 2012].

Impaired myocardial perfiasion reserve index (MPRI) associated with microvascular dysfianction and fibrosis occurs even prior to the onset of overt cardiomyopathy. Consistent with impaired mitochondrial respiratory chain fianction that leads to energy deficit, phosphorus magnetic resonance spectroscopy shows d ATP production in patient heart, which strongly correlates with the degree of cardiac hypertrophy. Cardiac dysfilnction, predisposing to congestive heart failure and supraventricular arrhythmias, is the primary mode of death in ~60% of patients with FRDA.

Although the fianction of frataxin is still under investigation, available ces support a role as an activator of iron-sulfilr (Fe-S) cluster biogenesis in mitochondria [C. L.

Tsai et al., 2010 and Schmucker et al., 2012]. In particular, frataxin was recently shown to control iron delivery for de novo Fe-S cluster biogenesis through activation of ne desulfurase activity [Colin et al., 2013].

Fe-S rs are essential prosthetic groups for many proteins with a y of functions and subcellular localizations. Frataxin deficiency leads to impairment of Fe-S cluster-containing ns, altered ar iron metabolism, mitochondrial ction and increased sensitivity to oxidative stress. Most mitochondrial and biochemical defects identified in human patients have been recapitulated in mouse models of FRDA [H. Puccio et al., 2001 and Simon et al. et al., 2004], providing valuable models for testing potential therapeutic interventions. Particularly, the MCK conditional mouse model, with complete frataxin deletion in cardiac and skeletal muscle, recapitulates the cardiomyopathy observed in FRDA patients with a more rapidly progressive course [H. Puccio et al., 2001 and H. Seznec et al., 2004]. rmore, the MCK mouse demonstrated that rophy and mitochondrial Fe-S cluster protein defects precede mitochondrial iron accumulation and increase sensitivity to oxidative stress.

To date, no treatment exists for stopping or slowing down the cardiomyopathy of FRDA. Current therapeutic approaches in clinical use or under evaluation are directed at alleviating symptoms and maximizing quality of life [R. B. Wilson 2012]. Thus, there is an important need for a novel therapeutic approach to treat cardiomyopathy associated with Friedreich ataxia.

SUMMARY OF THE INVENTION: In the present invention, the therapeutic effect of an AVVrh10 vector carrying a human frataxin cDNA on the c phenotype in a mammalian model of FRDA cardiomyopathy was investigated. The results showed that delivery of a vector encoding hFXN resulted in i) prevention of the development of e symptoms in omatic individuals and ii) reversal of disease symptoms in individuals who already ted cardiomyopathy, mitochondrial dysfunction and the biochemical impairment associated with frataxin deficiency. Of particular note is that the inventors showed that ry of a vector encoding hFXN resulted in a rapid reversal of the mitochondrial abnormalities leading to arrest of the cardiac fibrosis, restoration of the cardiac contractile apparatus and complete restoration of c on.

In a particular aspect, the present invention provides a use of an adeno-associated virus rh10 (AAVrh10) vector in the preparation of a medicament for preventing or treating [FOLLOWED BY PAGE 2a] - 2a - cardiomyopathy associated with Friedreich ataxia in a human, wherein the AAVrh10 vector ses a c acid sequence of a frataxin (FXN)-encoding nucleic acid or a fragment thereof, and wherein the AAVrh10 vector is formulated for administration to the subject at a dose of about 5 x 1012 viral genomes (vg)/kg.

In another particular aspect, the present invention provides a pharmaceutical composition for preventing or treating cardiomyopathy associated with Friedreich ataxia in a human in need thereof, comprising an adeno-associated virus rh10 (AAVrh10) vector sing a nucleic acid encoding a frataxin (FXN) or a fragment thereof and a ceutically acceptable vehicle, wherein the AAVrh10 vector is formulated for administration to the t at a dose of about 5 x 1012 viral genomes (vg)/kg.

More generally, the inventors show that by restoring an essential mitochondrial function with the use of the nuclear-encoded FXN gene, and y increasing the energy [FOLLOWED BY PAGE 3] _ 3 _ W0 2014/118346 production of the mitochondria, the myocardium on can be restored and the interstitial cardiac fibrosis stopped. Considering that inefficient energy ation and increased energy demand by the sarcomere have been suggested as a key consequence of many, if not all, hypertrophic myopathy associated mutations (Sweeney HL et al., 1998), they results demonstrate that the use of a gene that can restore energy failure may be usefill for preventing or treating cardiomyopathy linked to energy failure.

Thus, the invention s a method for preventing or ng cardiomyopathy due to energy failure in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a vector which comprises a nucleic acid sequence of a gene that can restore energy failure.

More particularly, the invention relates to a method for preventing or treating a cardiomyopathy associated with Friedreich ataxia in a subject in need thereof, comprising administering to said subject of a therapeutically effective amount of a vector which comprises a frataxin (FXN) encoding nucleic acid.

ED DESCRIPTION OF THE INVENTION: s of the invention A first object of the ion relates a method for ting or treating cardiomyopathy due to energy failure in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a vector which comprises a nucleic acid sequence of a gene that can restore energy failure.

As used herein the term omyopathy due to energy failure3, denotes a deterioration of the on of the myocardium leading to heart failure, cardiac remodelling, long-term high blood re, heart valve problems, impaired calcium cycling sensitivity, disturbed biochemical stress sensing, altered energy homeostasis due but not limited to mitochondrial dysfianction and fibrosis.

In a particular embodiment, the cardiomyopathy due to energy failure may be a dilated cardiomyopathy, a hypertrophic cardiomyopathy, a restrictive cardiomyopathy or an ischemic cardiomyopathy. _ 4 _ In another particular embodiment, the cardiomyopathy due to energy failure may be a cardiomyopathy due to a deficiency of fatty oxidation, including but not limited to y camitine deficiency, LCHAD, translocase, VLCAD.

In another particular embodiment, the cardiomyopathy due to energy failure may be a cardiomyopathy associated with Friedreich .

As used herein, the term "a gene that can restore energy failure" denotes a nuclear or mitochondrial gene that can restore energy failure and/or mitochondrial dysfilnction.

In a particular embodiment, the gene that can restore energy failure may be a nuclear gene ng a subunit of te dehydrogenase complex, a nuclear or a mitochondrial gene coding for a subunit of Complex 1, III, IV or V involved in the oxidative phosphorylation; a mitochondrial gene encoding transfer RNA, a gene involved in the esis of mitochondria such as SIRTl, a gene ed in the fusion of mitochondria such as OPAl, a gene involved in the fission of mitochondria such as FISl or a gene involved in the oxidation of fatty acid such as the very long-chain c acyl-CoA dehydrogenase.

In a particular embodiment, the gene that can restore energy failure is the in (FXN) gene.

As used herein in its broadest meaning, the term "preventing" or "prevention" refers to preventing the disease or condition from occurring in a subject which has not yet been sed as having it or which does not have any clinical symptoms.

As used herein, the term "treating" or "treatment", as used herein, means reversing, alleviating, or inhibiting the progress of the disorder or condition to which such term applies, or one or more symptoms of such er or condition. A "therapeutically effective amount" is intended for a minimal amount of active agent which is necessary to impart therapeutic benefit to a subject. For example, a "therapeutically effective " to a patient is such an amount which induces, ameliorates, ises, slows down the progression or otherwise causes an improvement in the ogical symptoms, disease progression or physiological conditions associated with or ance to succumbing to a disorder.

As used herein, the term "subject" denotes a mammal, such as a rodent, a feline, a canine, and a primate. Preferably a subject according to the invention is a human. In the context of the present invention, a "subject in need thereof" denotes a subject, ably a human, with a cardiomyopathy due to energy failure and more particularly a subject with a _ 5 _ W0 2014/118346 cardiomyopathy associated with eich ataxia. Subject with a cardiomyopathy associated with Friedreich ataxia presents some cardiac ms which may be, but are not limited to, a decrease of ejection on, increase of ventricular mass or cardiac hypertrophy. Thus, the method of the invention will be very useful to treat subject with such disease reich ataxia) ting such symptoms.

As used herein, the term "gene" refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular polypeptide or protein after being transcribed and translated.

As used herein, the terms "coding sequence", "a sequence which encodes a particular protein" or "encoding nucleic acid", denotes a nucleic acid sequence which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in viva when placed under the control of appropriate regulatory sequences. The ries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or otic DNA, and even synthetic DNA sequences.

In a particular embodiment, the invention relates to a method for preventing or treating a cardiomyopathy associated with Friedreich ataxia in a t in need thereof, comprising administering to said subject a therapeutically ive amount of a vector which comprises a frataxin (FXN) encoding nucleic acid.

In a particular embodiment, the invention relates to a method for treating a cardiomyopathy associated with Friedreich ataxia in a subject in need thereof, sing administering to said subject a therapeutically effective amount of a vector which comprises a frataxin (FXN) encoding c acid.

In a particular embodiment, the invention s to a method for reversing or stabilizing symptoms of cardiomyopathy associated with Friedreich ataxia in a subject in need thereof, comprising stering to said subject a therapeutically effective amount of a vector which comprises a frataxin (FXN) encoding c acid.

As used herein, the term "reversing symptoms of cardiomyopathy associated with Friedreich ataxia" denotes the restoration of cardiac function by, for example, improving ejection fraction and/or decreasing the ventricular mass in a subject in need thereof In a particular embodiment, the invention relates to a method for ing the dysfiJnction of cardiac mitochondria associated with Friedreich ataxia in a subject in need f, comprising administering to said subject a eutically effective amount of a vector which comprises a frataxin (FXN) ng nucleic acid.

In a particular embodiment, the invention s to a method for improving the cardiac mitochondria ated with Friedreich ataxia in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a vector which comprises a frataxin (FXN) encoding nucleic acid.

In a particular embodiment, the invention relates to a method for restoring cardiac filnction in a subject suffering of a cardiomyopathy associated with Friedreich ataxia comprising administering to said subject of a therapeutically effective amount of a vector which comprises a frataxin (FXN) ng nucleic acid.

In a particular embodiment, the invention relates to a method for improving cardiac filnction in a subject suffering of a cardiomyopathy associated with Friedreich ataxia sing administering to said subject a therapeutically effective amount of a vector which comprises a in (FXN) encoding nucleic acid.

In a particular embodiment, the invention relates to a method for treating a cardiomyopathy associated with Friedreich ataxia in an asymptomatic or pre-symptomatic subject in need thereof, comprising administering to said subject a therapeutically effective amount of a vector which comprises a frataxin (FXN) encoding nucleic acid.

In r particular embodiment, the invention relates to a method for treating a cardiomyopathy associated with Friedreich ataxia in a symptomatic t in need thereof, comprising administering to said subject a therapeutically ive amount of a vector which comprises a frataxin (FXN) encoding nucleic acid.

As used herein, the terms tomatic" or "pre-symptomatic" denotes a subject with the disease (Friedreich ) as defined by a genetic diagnosis (see for review Lynch DR et al., 2002) but with no clinical cardiac symptom.

As used herein, the terms symptomatic denotes a subject with the e (Friedreich ataxia) as defined by a genetic diagnosis and with the presence of cardiac symptoms ac hypertrophy, fibrosis, decreased myocardiac perfiJsion reserve index, impaired cardiac or _ 7 _ skeletal muscle mitochondrial respiratory chain fianction, subclinical cardiomyopathy, supraventricular arrhythmias, heart failure, systolic left ventricular dysfunction, fatigue. . .).

The FXN gene s the protein frataxin. This in is a protein localized to the mitochondrion. The frataxin is involved in assembly of iron-sulfilr clusters by regulating iron entry and the activity of the cysteine desulfiarase. A cDNA sequence for human FXN (transcript variant 1) is disclosed in Genbank Access Number NM_000144 or NG_008845 (SEQ ID NO: 1). The amino acid sequence of human frataxin is shown in SEQ ID N02.

The sequence ofthe c acid ofthe frataxin (cDNA) is (SEQ ID NO: 1): agtctccctt gggtcagggg tcctggttgc actccgtgct ttgcacaaag ctcc atttttgtta aatgcacgaa taag ctgggaagtt cttcctgagg tctaacctct agctgctccc ccacagaaga gtgcctgcgg ccagtggcca ccaggggtcg ccgcagcacc cagcgctgga gggcggagcg ggcggcagac ccggagcagc atgtggactc tcgggcgccg cgcagtagcc ggcctcctgg cgtcacccag cccagcccag gcccagaccc tcacccgggt cccgcggccg gcagagttgg ccccactctg ccgt ggcctgcgca tcga tgcgacctgc acgccccgcc gcgcaagttc gaaccaacgt ggcctcaacc agatttggaa tgtcaaaaag cagagtgtct atttgatgaa tttgaggaaa tctggaactt tgggccaccc aggctctcta gatgagacca cctatgaaag actagcagag gaaacgctgg actctttagc agagtttttt gaagaccttg cagacaagcc atacacgttt gaggactatg atgtctcctt tgggagtggt gtcttaactg tcaaactggg tggagatcta ggaacctatg tgatcaacaa gcagacgcca aacaagcaaa tctggctatc ttctccatcc agtggaccta agcgttatga ctggactggg aaaaactggg ccca cgacggcgtg tccctccatg agctgctggc cgcagagctc actaaagcct taaaaaccaa actggacttg ttgg cctattccgg aaaagatgct tgatgcccag ccccgtttta aggacattaa aagctatcag gccaagaccc cagcttcatt atgcagctga ggtctgtttt ttgttgttgt tgttgtttat tttttttatt cctgcttttg aggacagttg ggctatgtgt cacagctctg tagaaagaat gtgttgcctc ctaccttgcc cccaagttct taat ttctatggaa gattttttgg ggat ttcctccctc acatgatacc ccttatcttt tataatgtct tatgcctata tata acaaccttta aaaaagcaaa ataataagaa attc ggaa aatgaattgt cttcactctt ttga aggatttact gcaagaagta catgaagagc agctggtcaa cctgctcact gttctatctc agac acattaaagg gtagcctaca aatgttttca tttc aaagtgtaag cacttctgag ctctttagca ttgaagtgtc gaaagcaact cacacgggaa gatcatttct tatttgtgct ctgtgactgc gtgg cctgcactgg gttgtccagg gagacctagt gctgtttctc ccacatattc acatacgtgt gtat atatattttt tcaatttaaa ggttagtatg gaatcagctg ctacaagaat gcaaaaaat agac aagaaaagag gaaaaaaagc cgttttcatg agctgagtga tgtagcgtaa caaacaaaat catggagctg aggaggtgcc ttgtaaacat gaaggggcag ataaaggaag gagatactca tgttgataaa gagagccctg gtcctagaca agcc acaaagtagt tgtccctttg tggacaagtt tcccaaattc cctggacctc tgcttcccca tctgttaaat gagagaatag agtatggttg attcccagca ttcagtggtc ctgtcaagca acctaacag _ 8 _ W0 18346 ctagttctaa ttccctattg ggtagatgag gggatgacaa agaacagttt ttaagctata taggaaacat tgttattggt gttgccctat cgtgatttca gttgaattca tgtgaaaata atagccatcc ttggcctggc gcggtggctc acacctgtaa tcccagcact tttggaggcc ggtg gatcacctga ggtcaggagt tcaagaccag cctggccaac atgatgaaa cccgtctcta ctaaaaatac aaaaaattag ccgggcatga tggcaggtgc ctgtaatccc agctacttgg gaggctgaag cggaagaatc gcttgaaccc agaggtggag gttgcagtga gccgagatcg tgccattgca ctgtaacctg ggtgactgag caaaactctg tctcaaaata ataataacaa tataataata ataatagcca attg tacccttact gggttaatcg tattatacca cattacctca ttttaatttt tactgacctg cactttatac aaagcaacaa gcctccagga cattaaaatt catgcaaagt tatgctcatg ttatattatt ttcttactta aagaaggatt tggc tgggcatggt ggcgtgcacc tgtaatccca ggtactcagg aggctgagac gggagaattg cttgacccca ggcggaggag gttacagtga gtcgagatcg tacctgagcg acagagcgag actccgtctc aaaaaaaaaa aaaaggaggg tttattaatg agaagtttgt attaatatgt agcaaaggct tttccaatgg gtgaataaaa acacattcca ttaagtcaag ctgggagcag tggcatatac ctatagtccc agctgcacag gaggctgaga caggaggatt gcttgaagcc aggaattgga gatcagcctg ggcaacacag caagatccta tctcttaaaa aaagaaaaaa aaacctatta ataataaaac agtataaaca aaagctaaat aggtaaaata ttttttctga aataaaatta ttttttgagt ctgatggaaa tgtttaagtg cagtaggcca gtgccagtga gaaaataaat aacatcatac atgtttgtat gtgtttgcat cttgcttcta ctgaaagttt cagtgcaccc cacttactta gaactcggtg acatgatgta ctcctttatc acag cacaaaagag gtatgcagtg ctct gacatgaaag ttaa ggaatctggg ctcttatggg gtccttgtgg gccagccctt caggcctatt ttactttcat tata attg gtttgattat ctcgttccca aggcagtggg agatccccat ttaaggaaag aaaaggggcc agtg gctcatgcct gtaatcccag ggga ggctgaggca agtgtatcac ctgaggtcag gagttcaaga ccagcctggc caacatggca aaatcccgtc tctactaaaa atattaaaaa attggctggg cgtggtggtt cgtgcctata atttcagcta aggc tgaggcagga gaatcgctgt aacctggggg gtggaggttg cagtgagacg agatcatgcc acttcactcc agcctggcca acagagcca actccgtctc aaataaataa ataaataaat aaagggactt caaacacatg aacagcagcc aggggaagaa tcaaaatcat attctgtcaa tgga aaagtaccac tgtgtgtacc aatagcctcc ccaccacaga ccctgggagc atcgcctcat ttatggtgtg gtccagtcat gaag gatgagtttc caggaaaagg ttattaaata ttcactgtaa catactggag gaggtgagga attgcataat acaatcttag aaaacttttt cttt ctattttttg agacaggatc tcactttggc actcaggctg gaggacagtg gtacaatcaa agctcatggc agcctcgacc tccctgggct tgggcaatcc tcccacaggt gtgcacctcc atagctggct aatttgtgta ttttttgtag agatggggtt tcaccatgtt gcccaggctg gtctctaaca cttaggctca agtgatccac cgtc ctcccaagat gctgggatta caggtgtgtg ccacaggtgt tcatcagaaa gctttttcta ttatttttac cttcttgagt gggtagaacc acat taaa atgttctggc atgacttatt tagctctctg gaattacaaa gaaggaatga ggtgtgtaaa agagaacctg ggtttttgaa attt taat tctg cctcttactt gtttgtagac actgacagtg gcctcatgtt tttttttttt ttaatctata aaatggagat atctaacatg ttgagcctgg gcccacaggc aaagcacaat cctgatgtga gaagtactca gttcatgaca actgttgttc tcacatgcat attt catattcaca ttggaggact tctcccaaaa tatggatgac gttccctact caaccttgaa cttaatcaaa atactcagtt tacttaactt cgtattagat tccc catt tatcgtgtgc atgc ttatatttta cttgatcttt tgcatacctt ctaaaactat tttagccaat ttaaaatttg acagtttgca _ 9 _ W0 2014/118346 ttaaattata caat atgctttatc cagctatacc tgccccaaat tctgacagat gcttttgcca cctctaaagg aagacccatg ttcatagtga tggagtttgt gtggactaac catgcaaggt tgccaaggaa aaatcgcttt acgcttccaa ggtacacact aagatgaaag taattttagt ccgtgtccag ttggattctt tagt tatcttctgc tagaacaaac taaaacagct cagc aagggagaaa ggggaaggag gggcaaagtt ttgaaatttc atgtaaattt atgctgttca aaacgacgag ttcatgactt tgtgtataga gtaagaaatg ccttttcttt tttgagacag agtcttgctc tgtcacccag gctggagtgc agtggcacga tctgggctca ctacaacctc cgcctcctgg gttcaagcaa ttctctgcct cagcctcccg agtagctggg attacaggtg cctgccacca cacccggcta atttttgtat ttttagtaga gacggggttt caccatcatg gccaggctgg tcttgaactc ctgacctagt aatccacctg cctccgcctc ccaaagtgct acag gcgtgagcca ctgcacccag ccagaaatgc cttctaatct tatc ttaattagcc aggacacttg atcc cgaagtacct gatcagtggc ccctttggaa tgtgtaaaac tcagctcact tatatccctg catccgctac agagacagaa tccaagctca tatgttccat cttctctggc tgtatagttt aaggaatgga aggcaccaga acagatttat tgaaatgttt attagctgaa gatttattta gacagttgag gaaaacatca gcacccagca gtaaaattgg ctctcaaaga ttttcttctc ctgtggaaag tcagacctct gaggccccat ccaggtagaa gtactagtgc ggcc tctgctgtcc acttgtgttt tctg tgggaacatt gttaacgcca catcttgacc tcaaattgtt tagctcctgg ccagacacgg tggctcacac ctgtaatccc agcactttga gaggctgagg caggtggatc ggtt aggagttcga ggccagcctg gtcaacatgg taaaaccccg cctctactaa aaaa attagctggc cgtagtggcg cacgcctgtt atcccagcta ctcgggaggc agga gaattgcttg aacctgggtg gtggaggttg cagtgagccg agattacacc actgcactcc agcctgggtg acaagaggga atta aaaaaatgta attcccgtgt ctgccatctt aagtgtaaag gtggctaaat tatatagaaa aataagacaa ttcc caattacatt cctttcctac cgcactctat gatgctagct gagatttttc caaaagaaaa aaat aaaacccta gagaaagaaa aactttaaat ccctccaaag agta atagaaacag atgagtttgg gatt tctctgtaag attgcctagg ctgtgtactg cacatctcca actg ttgacagaga ttataactac aatgtgaagt gaatggtgcc actgacagtt atgcaaaccg tccagagcat agccacctga tggg attcctcttg ccagtccatc agcagttccc cttgaaagtt tcaccaaaca tcccttaaat tctc ctgcccgtcc gagg tcctcatcat cctg catttttgca ggagctttct tatatccacc ttcctccttt tctctcagcc catcatctag ctacacagtc tccagggtaa gctttcagaa aggcaatctc ttgtctgtaa aacctaagca ggaccaaggc tctt aaaa atgtgctttt ctgactgaac tgttcaggca ctgactctac atataattat gcttttctac cccctcacac tcaacacttt gactccagca atcccaaatc cccagatccc taagtgtgct gtgctatttt cacgtggctc tcagacttgg ctgt ttccattttg gtctttattc cccacatctc tgcctggggg gtagattcta ccctgaaaaa tgttcttggc acagccttgc aaactcctcc tccactcagc ctctgcctgg atgcccttga ttgttccatg tcctcagcat accatgtttg tctttcccag cactgaccta ccatgtgtca cccctgcttg gctgtacctt ccatgaggct aggactatgt gtctcctttg ttgactgctg ttgccctagc atcttgcaca gttccttgca cacaattaga gctctataaa tgtcaaataa atgtgttata attatatgtt taagatagtt gttcaaataa actctaaata accccaac.

The sequence of the frataxin protein is (SEQ ID NO:2): MWTLGRRAVAGLLASPSPAQAQTLTRVPRPAELAPLCGRRGLRTDIDATCTPRRASS _ 10 _ QIWNVKKQSVYLMNLRKSGTLGHPGSLDETTYERLAEETLDSLAEFFEDLA DKPYTFEDYDVSFGSGVLTVKLGGDLGTYVINKQTPNKQIWLSSPSSGPKRYDWTGK NWVYSHDGVSLHELLAAELTKALKTKLDLSSLAYSGKDA.

In a ular embodiment, the invention provides a c acid construct comprising sequence SEQ ID NO:l or a variant thereof for treating cardiomyopathy associated with Friedreich ataxia.

The variants include, for instance, naturally-occurring variants due to allelic variations between individuals (e.g., polymorphisms), alternative splicing forms, in particular transcript variants 2 and 3 (accession numbers NM_OOl 161706 and NM_181425), etc. The term variant also includes FXN gene sequences from other sources or organisms. Variants are ably substantially homologous to SEQ ID NO:1, i.e., exhibit a nucleotide sequence identity of typically at least about 75%, preferably at least about 85%, more preferably at least about 90%, more preferably at least about 95%, 96%, 97%, 98%, or 99% with SEQ ID NO: 1.

Variants of a FXN gene also include c acid sequences, which hybridize to a sequence as defined above (or a mentary strand thereof) under stringent hybridization conditions.

Typical stringent hybridisation conditions include temperatures above 30° C, preferably above 35°C, more ably in excess of 42°C, and/or salinity of less than about 500 mM, preferably less than 200 mM. Hybridization conditions may be adjusted by the skilled person by modifying the temperature, salinity and/or the concentration of other reagents such as SDS, SSC, etc.

In a particular embodiment, the FXN-encoding nucleic acid is a fragment of the SEQ ID NO:l which encodes for the amino acid sequence 81-210 of the SEQ ID NO:2 (named variant 0") or a variant f, "variant" having the meaning provided above with respect to nucleotide sequence identity and hybridization.

In a another particular embodiment, a sequence known as mitochondrion-targeting signal or mitochondrial ing signal may be added to the FXN-encoding sequence or variant thereof, including, for e the FXN-encoding sequence "81-210". Sequences known as mitochondrion-targeting signal or mitochondrial targeting signal are referred to as MTS by the skilled person.

A MTS sequence can be identified within a protein or nucleic acid ce by a person of ordinary skill in the art.

Most mitochondrion-targeting peptides t of a N—terminal pre-sequence of about to 100 residues, preferably of about 20 to 80 residues. They are enriched in arginine, _ 11 _ leucine, serine and alanine. Mitochondrial pre-sequences show a statistical bias of positively charged amino acid residues, provided mostly through arginine residues; very few sequences contain negatively charged amino acids. Mitochondrion-targeting peptides also share an ability to form an hilic alpha-helix.

A complete description of a method to identify a MTS is available in: MG. Claros, P.

Vincens, 1996 (Eur. J. Biochem. 241, 779-786 (1996), "Computational method to predict mitochondrially imported proteins and their targeting sequences"), the content of which is herein incorporated by reference.

In another embodiment, the invention s to a method for use in the prevention or treatment of es associated with frataxin deficiency in a subject in need therefore, comprising to said subject administering a therapeutically effective amount of a vector which comprises a nucleic acid encoding frataxin.

In r embodiment, the invention relates a method for use in the prevention or treatment of cardiomyopathy due but not limited to energy e in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a vector which comprises a nucleic acid sequence of a gene that can e energy e.

In another particular embodiment, the invention s a method for use in the prevention or treatment of cardiomyopathy due but not limited to energy failure in a subject in need thereof, comprising administering to said t a eutically effective amount of a vector which ses a frataxin (FXN) encoding nucleic acid.

The invention also relates to a vector which comprises a nucleic acid sequence of a gene that can restore energy failure for use in treatment or prevention of cardiomyopathy due to energy failure in a subject in need thereof In a particular embodiment, the invention relates to a vector which comprises a frataxin (FXN) ng nucleic acid for use in the ent or prevention of a cardiomyopathy associated with Friedreich ataxia in a subject in need thereof In a particular ment, the invention relates to a vector which comprises a frataxin (FXN) encoding nucleic acid for use in the treatment of a cardiomyopathy associated with Friedreich ataxia in a subject in need thereof _ 12 _ In a ular embodiment, the invention relates to a vector which comprises a frataxin (FXN) encoding nucleic acid for ing or stabilizing symptoms of cardiomyopathy associated with Friedreich ataxia in a subject in need thereof.

In a particular embodiment, the invention relates to a vector which comprises a frataxin (FXN) encoding nucleic acid for reversing the dysfianction of cardiac mitochondria associated with Friedreich ataxia in a subject in need thereof.

In a ular embodiment, the invention s to a vector which comprises a frataxin (FXN) ng nucleic acid for improving the cardiac mitochondria associated with Friedreich ataxia in a subject in need thereof.

In a particular embodiment, the invention relates to a vector which comprises a frataxin (FXN) encoding nucleic acid for restoring c fianction in a subject suffering of a cardiomyopathy associated with Friedreich ataxia.

In a particular embodiment, the invention relates to a vector which comprises a frataxin (FXN) encoding nucleic acid for improving c fianction in a subject suffering of a cardiomyopathy associated with Friedreich ataxia.

In a particular embodiment, the invention relates to a vector which ses a frataxin (FXN) encoding nucleic acid for use in the treatment a cardiomyopathy associated with Friedreich ataxia in an asymptomatic or pre-symptomatic t in need thereof In a particular embodiment, the invention relates to a vector which ses a in (FXN) encoding nucleic acid for use in the treatment a cardiomyopathy associated with Friedreich ataxia in a symptomatic subject in need f Non viral vectors In a particular embodiment, the vector use according to the invention is a non viral vector. Typically, the non viral vector may be a plasmid which includes nucleic acid sequences encoding FXN gene, or variants thereof, as described above.

The Viral vectors _ 13 _ W0 2014/118346 2014/051966 Gene delivery viral vectors useful in the practice of the present invention can be constructed utilizing methodologies well known in the art of molecular biology. Typically, viral vectors carrying transgenes are assembled from polynucleotides encoding the transgene, suitable regulatory elements and elements necessary for production of viral ns which mediate cell transduction.

The terms "Gene transfer" or "gene delivery" refer to methods or systems for reliably inserting foreign DNA into host cells. Such methods can result in transient expression of non integrated transferred DNA, extrachromosomal ation and expression of transferred replicons (e. g. episomes), or integration of transferred genetic material into the genomic DNA of host cells.

Examples of viral vector include but are not limited to iral, retroviral, lentiviral, herpesvirus and adeno-associated virus (AAV) vectors.

Such recombinant viruses may be ed by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses.

Typical examples of virus packaging cells include PA3 17 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-defective inant viruses may be found for instance in WO95/l4785, WO96/22378, USS,882,877, US6,0l3,Sl6, US4,86l,7l9, USS,278,056 and WO94/l9478.

In one embodiment, adeno-associated viral (AAV) s are employed.

In other embodiments, the AAV vector is AAVl, AAV2, AAV3, AAV4, AA5, AAV6, AAV7, AAV8, AAV9, AAVrth or any other serotypes of AAV that can infect humans, monkeys or other s.

In an exemplary embodiment, the AAV vector is an AAVrth.

By an "AAV vector" is meant a vector derived from an associated virus serotype, including without limitation, AAV-l, AAV-2, AAV-3, AAV-4, AAV-5, AAV6, etc.

AAV s can have one or more of the AAV wild-type genes deleted in whole or part, preferably the rep and/or cap genes, but retain fianctional flanking ITR sequences. Functional ITR sequences are necessary for the rescue, replication and packaging of the AAV virion.

Thus, an AAV vector is defined herein to e at least those sequences required in cis for replication and packaging (e. g., fianctional ITRs) of the virus. The ITRs need not be the wild- type tide sequences, and may be altered, e. g by the insertion, deletion or substitution of nucleotides, so long as the sequences provide for fianctional , replication and packaging.

AAV expression vectors are constructed using known techniques to at least provide as ively linked components in the direction of transcription, control elements including a _ 14 _ W0 18346 transcriptional initiation , the DNA of interest (i.e. the FXN gene) and a transcriptional termination region.

The control elements are selected to be fianctional in a mammalian cell. The resulting construct which contains the operatively linked components is bounded (5' and 3’) with functional AAV ITR sequences. By "adeno-associated virus ed terminal repeats " or "AAVITRs" is meant the cognized regions found at each end ofthe AAV genome which fianction together in cis as origins of DNA replication and as packaging signals for the virus.

AAV ITRs, together with the AAV rep coding region, provide for the efficient excision and rescue from, and integration of a tide sequence interposed between two flanking ITRs into a ian cell genome. The nucleotide sequences of AAV ITR regions are known.

See, e.g., Kotin, 1994; Berns, KI "Parvoviridae and their ation" in Fundamental Virology, 2nd Edition, (B. N. Fields and D. M. Knipe, eds. ) for the AAV-2 sequence. As used herein, an "AAV ITR" does not necessarily comprise the wild-type nucleotide sequence, but may be altered, e. g., by the insertion, deletion or substitution of nucleotides. Additionally, the AAV ITR may be derived from any of several AAV serotypes, including without tion, AAV-l, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, etc. Furthermore, 5' and 3' ITRs which flank a selected nucleotide sequence in an AAV vector need not necessarily be cal or derived from the same AAV serotype or isolate, so long as they fianction as intended, i.e., to allow for on and rescue of the sequence of st from a host cell genome or vector, and to allow integration of the heterologous sequence into the recipient cell genome when AAV Rep gene products are present in the cell. Additionally, AAV ITRs may be derived from any of several AAV serotypes, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV 5, AAV-6, etc. Furthermore, 5 'and 3' ITRs which flank a selected nucleotide sequence in an AAV expression vector need not arily be identical or derived from the same AAV serotype or isolate, so long as they function as intended, i. e., to allow for excision and rescue of the sequence of interest from a host cell genome or vector, and to allow integration of the DNA molecule into the recipient cell genome when AAV Rep gene products are present in the cell.

Particularly preferred are vectors derived from AAV serotypes having tropism for and high transduction efficiencies in cells of the mammalian myocardium, particularly cardiomyocytes and myocyte itors. A review and ison of transduction efficiencies of different serotypes is provided in Cearley CN et al., 2008. In other non-limiting examples, preferred vectors include vectors derived from any serotypes like AAVl, AAV2, _ 15 _ W0 2014/118346 AAV3, AAV4, AA5, AAV6, AAV7, AAV8, AAV9, or AAVrth, which have also been shown to transduce cells of cardiomyocytes.

The selected nucleotide ce is operably linked to control elements that direct the transcription or expression thereof in the subject in viva. Such control elements can comprise control sequences ly ated with the selected gene.

Alternatively, heterologous control ces can be employed. Useful logous control sequences generally include those derived from sequences encoding mammalian or viral genes. Examples include, but are not limited to, the phophoglycerate kinase (PKG) er, CAG, MCK (muscle creatine ), the SV40 early promoter, mouse mammary tumor virus LTR promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a galovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), rous sarcoma virus (RSV) promoter, synthetic promoters, hybrid promoters, and the like. The promoters can be of human origin or from other species, including from mice. In addition, sequences derived from nonviral genes, such as the murine metallothionein gene, will also find use herein. Such promoter sequences are commercially available from, e. g. Stratagene (San Diego, CA).

Examples of heterologous promoters include the CMV promoter.

Examples of inducible promoters include DNA responsive elements for ecdysone, tetracycline, hypoxia andauf1n.

The AAV expression vector which harbors the DNA molecule of st bounded by AAV ITRs, can be constructed by directly inserting the selected sequence (s) into an AAV genome which has had the major AAV open reading frames ("ORFs") excised rom.

Other portions of the AAV genome can also be deleted, so long as a ient portion of the ITRs remain to allow for replication and packaging ons. Such constructs can be designed using techniques well known in the art. See, e. g. U. S. Patents Nos. 5,173, 414 and ,139, 941; International Publications Nos. WO 92/01070 (published 23 January 1992) and WO 93/03769 (published 4 March 1993); Lebkowski et al., 1988 ; Vincent et al., 1990; Carter, 1992 ; ka, 1992 ; 1994; ng and Smith, 1994 ; and Zhou et al., 1994. Alternatively, AAV ITRs can be excised from the viral genome or from an AAV vector containing the same and fiJsed 5' and 3' of a selected nucleic acid construct that is present in another vector using standard ligation techniques. AAV vectors which contain ITRs have been described in, e. g. U. S. Patent no. 5,139, 941. In particular, several AAV vectors are described therein which are available from the American Type Culture Collection ("ATCC") _ 16 _ W0 2014/118346 under Accession Numbers 53222,53223, 53224,.53225 and 53226. Additionally, chimeric genes can be produced synthetically to include AAV ITR ces ed 5' and 3' of one or more selected nucleic acid sequences. red codons for expression of the chimeric gene sequence in mammalian CNS cells can be used. The complete chimeric sequence is assembled from overlapping oligonucleotides prepared by standard methods. See, e. g., Edge, 1981 ; Nambair et al., 1984 ; Jay et al., 1984. In order to produce AAV virions, an AAV expression vector is introduced into a suitable host cell using known techniques, such as by transfection. A number of transfection techniques are generally known in the art. See, e. g. , Graham et al., 1973;, ok et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis etal. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al., 1981. Particularly suitable transfection s e calcium phosphate co-precipitation (Graham et al., 1973), direct microinjection into cultured cells (Capecchi, 1980), electroporation (Shigekawa et al., 1988), liposome ed gene transfer no et al., 1988), lipid-mediated transduction (Felgner et al., 1987), and nucleic acid delivery using high-velocity microprojectiles (Klein et al., 1987).

For instance, a red viral vector, such as the , comprises, in addition to a FXN encoding nucleic acid sequence, the backbone of AAV vector with ITR derived from AAV-Z, the promoter, such as the mouse PGK (phosphoglycerate kinase) gene or the cytomegalovirus/B-actin hybrid promoter (CAG) ting of the enhancer from the cytomegalovirus immediate gene, the promoter, splice donor and intron from the chicken [3- actin gene, the splice acceptor from rabbit B-globin, or any promoter such as PGK, CAG, MCK.

Delivery of the vectors It is herein provided a method for treating cardiomyopathy due to energy failure in a subject, said method sing: (a) providing a vector as defined above, which comprises a c acid sequence of a gene that can restore energy failure; and (b) delivering the vector to the subject in need thereof and whereby the gene is expressed by the transduced cells at a therapeutically effective level.

In a particular embodiment, it is herein provided a method for treating cardiomyopathy associated with Friedreich ataxia in a subject, said method comprising: _ 17 _ (a) providing a vector as defined above, which ses a FXN encoding c acid; and (b) delivering the vector to the t in need thereof and y FXN is expressed by the transduced cells at a therapeutically ive level.

In a particular method, the vector is delivered directly into the myocardium by epicardiac injection followed by minithoracotomy, by intracoronary injection, by endomyocardic injection, by subepicardial or epicardial injection or other type of injection useful in the heart.

Additional routes of administration may also comprise local application of the vector under direct visualization, e. g., superficial cortical ation, or other nonstereotactic application. The vector may be delivered intrathecally, in the cles or by enous injection.

The target cells of the vectors of the present ion are cells of the myocardium of a subject afflicted with a cardiomyopathy associated with Friedreich ataxia. Preferably the t is a human being, adult or child.

However the invention encompasses delivering the vector to biological models of the disease. In that case, the biological model may be any mammal at any stage of development at the time of delivery, e. g., embryonic, fetal, infantile, juvenile or adult. Furthermore, the target myocardium cells may be essentially from any source, especially any cells derived from hiPS from FRDA patients, an primates and mammals of the orders Rodenta (mice, rats, rabbit, hamsters), Camivora (cats, dogs), and Arteriodactyla (cows, pigs, sheep, goats, horses) as well as any other non-human system (e. g. zebrafish model system).

The vectors used herein may be formulated in any suitable vehicle for delivery. For instance they may be placed into a pharmaceutically acceptable suspension, solution or emulsion. le mediums include saline and liposomal preparations. More specifically, pharmaceutically acceptable carriers may include sterile aqueous of non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Intravenous vehicles include fluid and nutrient ishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. _ 18 _ W0 2014/118346 Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.

A colloidal dispersion system may also be used for targeted gene delivery. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

The red doses and regimen may be determined by a ian, and depend on the age, sex, weight, of the subject, and the stage of the disease. As an example, for delivery of a nucleic acid ce encoding an FXN polypeptide using a viral expression vector, each unit dosage of FXN polypeptide expressing vector may comprise 2.5 to 100 ul of a composition including a viral sion vector in a pharmaceutically acceptable fluid at a concentration ranging from 1011 tolO16 viral genome per ml for example.

The invention also relates to a vector which comprises a nucleic acid ce of a gene that can restore energy failure for use in treatment or prevention cardiomyopathy due to energy failure in a subject wherein the vector is delivering the subject in need thereof and wherein the gene is expressed by the transduced cells at a therapeutically effective level.

In a particular ment, the invention relates to a vector which comprises a FXN encoding nucleic acid for use in ent or prevention of cardiomyopathy associated with Friedreich ataxia in a subject n the vector is delivering the subject in need thereof and wherein FXN is sed by the transduced cells at a therapeutically effective level.

In a particular embodiment, the invention relates to a vector which comprises a FXN encoding nucleic acid for reversing symptoms of myopathy associated with eich ataxia in a t in need thereof wherein the vector is delivering the subject in need thereof and wherein FXN is expressed by the transduced cells at a therapeutically effective level.

Pharmaceutical composition A second object ofthe invention ns a pharmaceutical composition for preventing or treating cardiomyopathy due to energy failure in a subject in need f, which comprises a therapeutically effective amount of a vector which comprises a nucleic acid sequence of a gene that can restore energy failure. _ 19 _ In a particular embodiment, the invention concerns a pharmaceutical composition for preventing or treating cardiomyopathy associated with Friedreich ataxia in a subject in need thereof, which comprises a therapeutically effective amount of a vector which comprises a FXN encoding nucleic acid.

By a "therapeutically effective amount" is meant a sufficient amount of the vector of the invention to treat a cardiomyopathy ated with Friedreich ataxia at a reasonable benefit/risk ratio applicable to any medical treatment.

It will be understood that the single dosage or the total daily dosage of the compounds and compositions of the present invention will be d by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the c nd employed; the c composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of stration, and rate of excretion of the c compound employed; the on of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired eutic effect and to gradually increase the dosage until the d effect is achieved. However, the daily dosage of the products may be varied over a wide range per adult per day. The therapeutically effective amount of the vector according to the invention that should be administered, as well as the dosage for the treatment of a pathological ion with the number of viral or non-viral particles and/or pharmaceutical itions of the invention, will depend on us factors, including the age and condition of the patient, the severity of the disturbance or disorder, the method and frequency of administration and the particular peptide to be used.

The presentation of the pharmaceutical compositions that contain the vector according to the invention may be in any form that is suitable for the selected mode of administration, for example, for intraventricular, intramyocardium, intracoronary or intravenous administration.

In the pharmaceutical compositions of the present invention for intramuscular, intravenous, intramyocardium, intracoronary or intraventricular administration, the active principle, alone or in combination with another active principle, can be administered in a unit _ 20 _ W0 18346 administration form, as a mixture with conventional pharmaceutical supports, to s and human beings. ably, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or um phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.

The pharmaceutical forms suitable for able use include sterile aqueous solutions or dispersions; ations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fiangi.

Solutions comprising compounds of the ion as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid hylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of rganisms.

The vector according to the invention can be formulated into a composition in a l or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups ofthe protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be d from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric ides, and such organic bases as pylamine, hylamine, ine, procaine and the like.

The carrier can also be a solvent or sion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid hylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifilngal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many _ 21 _ W0 2014/118346 cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.

Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents ng absorption, for example, aluminium monostearate and Sterile inj ectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as ed, ed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile able solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient fiom a previously sterile-filtered solution thereof Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug e capsules and the like can also be employed. le doses can also be administered.

In another ment, the invention relates to a pharmaceutical composition for treating or preventing diseases associated with fiataxin deficiency in a t in need therefore, sing to said subject administering a therapeutically effective amount of a vector which comprises a nucleic acid encoding frataxin.

The invention will be filrther illustrated by the following figures and examples.

However, these examples and figures should not be interpreted in any way as limiting the scope ofthe present invention.

S: Figure 1. Administration of AAVrthCAG-hXN vector at 3 weeks of age prevents the onset of cardiac failure and rescues survival in mptomatic MCK mice at a dose of 5x1013 vg/kg. (A) Survival rates of wild-type (black solid line), treated (grey dotted line) and _ 22 _ untreated (black dotted line) MCK mice. n=9-lO for each group. (B) Relative quantification of atrial natriuretic peptide (ANP), brain natriuretic e (BNP) and sarcoplasmic reticulum Ca2+ ATPase 2a) mRNA expressions in heart at 8 and 35 weeks for wild- type (white), treated (grey) and untreated ) MCK mice; n=3-5 per group (*P<0.05; ***P<0.001). mRNA expression was normalized to 18S ribosomal RNA and is presented as a fold change relative to wild-type values. Data are represented as means :: SD.

Figure 2. Administration ofAAVrh10.CAG-hXN vector, at a dose of 5x1013 vg/kg, at 7 weeks of age in symptomatic MCK mice with severe cardiac failure reverses the cardiac contractile dysfianction, Fe-S cluster proteins, and cardiomyocyte and mitochondrial ultrastructure disorganization. (A) Longitudinal echocardiographic assessment of the left cle mass (LVM, left) and the shortening on (SF, right) for wild-type (black circles) mice, treated (light grey squares) and untreated (grey triangle) MCK mice. Data are represented as means :: SD. n=9-ll for each group. (B) Survival rates of wild-type mice (black solid line), treated (grey dotted line) and untreated (black dotted line) MCK mice. n=9- 11 for each group. (C) Relative quantification of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and sarcoplasmic reticulum Ca2+ ATPase (Serca2a) mRNA expressions in heart at 8, l5 and 22 weeks for wild-type ), treated (grey) and ted (black) MCK mice; n=3-5 per group and per age (*P<0.05; **P<0.0l; ns: statistically non- signif1cant). mRNA expression was normalized to 18S mal RNA and is presented as a fold change relative to wild-type values. Data are represented as means :: SD. (D) Biochemical measurements of combined cytosolic and mitochondrial aconitases (Aco) and succinate dehydrogenase (SDH, complex 11) activities in heart from wild-type (white) mice, treated (grey) and untreated (black) MCK mice at 8, 15 and 22 weeks of age; n=3-6 per group and per age (**P<0.0l). Isocitrate dehydrogenase (IDH) activity was used to normalize SDH and aconitase activites for total mitochondrial content.

Figure 3. Treatment of MCK mice at 5 weeks with 10 fold decreased dose of vector delays the onset of c failure. (a-d) Echocardiographic assessment of the left cle shortening fraction (a), ejection fraction (b), cardiac output (c) and left ventricle mass ed to body weight (d) for MCK mice treated with a dose of 5x1013 vg/kg ( or 5x1012 , n=3) vg/kg (0, n=3), as well as for control wild-type (*, n=4) and untreated MCK (O, n=10) mice. (e-g) Kinetic of individual echocardiographic assessment of left ventricular shortening on for MCK mice treated with 5x1013 vg/kg (e) or 5x1012 vg/kg (f and g). _ 23 _ Table 1: Echocardiographic parameters, percentage of SDH positive cardiomyocytes and vector genome copy per individual mice. Abbreviations: vg/kg, vector genome per kilogram; LV, left cle; SDH, Succinate dehydrogenase; ND, not determined. Values are presented as mean of experimental replicate of individual measure. 2014/051966 $38 32%. 38on an OQNMMSm $353 whfiom :3 was: 95385:"; >A ofiafiwomggoaom— 2:an ":35O 353.5 315225 E5295 36m— SB? awn? H 23%? HHE _ 25 _ W0 2014/118346 EXAMPLES: Example 1: Material & Methods Adeno-associated viral vector construction and production Human in (hFXN) cDNA, including the mitochondrial targeting sequence, filSGd to a HA tag was subcloned in a pAAV2-CAG d (Sondhi, Hackett et a1. 2007) to e pAAV2-CAG-hFXN that included the viral inverted al repeat (ITR) from AAV2; the cytomegalovirus/B-actin hybrid promoter, ting of the enhancer from the cytomegalo- virus immediate-early gene, the promoter, splice donor, and intron from the chicken B-actin gene, and the splice acceptor from rabbit B-globin. The AAVrh10.CAG- hFXN—HA vector was produced as described r (Rabinowitz, Rolling et a1. 2002) in the Vector Core at the University Hospital of Nantes (http://www.vectors.nantes.inserm.fr). The final titers of the two batches used were 5.4 xlO12 vg/m1 and 2.15 x1013 vg/m1, respectively.

Animal procedures Mice with a specific deletion of Fxn gene in cardiac and skeletal muscle (MCK-Cre- Fan3/L-) (MCK mice) in 100% C57BL/6J background were generated and genotyped as previously bed (Puccio, Simon et a1. 2001). Mice were maintained in a temperature and humidity controlled animal facility, with a 12 hours light/dark cycle and free access to water and a standard rodent chow (D03, SAFE, Villemoisson—sur-Orge, France). All animal procedures and experiments were approved by the local ethical committee for Animal Care and Use (Com’Eth 2011-07), and were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health). For biodistribution studies, three weeks old ype mice were anesthetized by eritoneal injection of ketamine/xylazine (75/10 mg/kg) to allow intravenous administration by retro-orbital injection of 0.CAG-FXN at a dose of 5X1013vg/kg, and sacrificed at 7 weeks of age (4 weeks post-injection). For gene therapy studies, three or seven weeks old MCK mice were anesthetized by intraperitoneal injection of ketamine/xylazine (75/10 mg/kg or 60/8 mg/kg, _ 26 _ W0 2014/118346 respectively) to allow intravenous administration by retro-orbital injection of AAVrh10.CAG- FXN at a dose of 5x1013vg/kg. Untreated MCK and WT mice littermates were injected with equivalent volume of saline solution. Survival was evaluated daily and mice weight weekly.

The mice cardiac fianction was evaluated under isofluorane anesthesia (1-2%) by echocardiography by an experimenter blinded to mice genotype and treatment regimen, as previously described (Seznec, Simon et al. 2004). Animals were killed by C02 inhalation at 8, 15, 22 or 35 weeks, and tissues samples for biochemical and molecular analysis were immediately frozen in liquid nitrogen. For histological analysis, mice were anesthetized by intraperitoneal injection of ketamine/xylazine and perfilsed with cooled saline solution. For ogical analysis of dorsal root ganglia, spinal cord and cardiac tissue was embedded in OCT Tissue Tek (Sakura Finetechnical, Torrance, rnia) and snap-frozen in isopentane chilled in liquid nitrogen. Samples of skeletal muscles were directly snap-frozen in isopentane chilled in liquid nitrogen. For electron microscopy analysis, small samples from the middle of left cle and its apex were collected, then fixed and ed in Epon as previously described (Puccio, Simon et al. 2001).

Histopathology, enzyme histochemistry and electron microscopy For histochemical analysis, 10um cryosections were stained either with xylin and eosin (H&E), Sirius red and Fast green to label extracellular collagen, or DAB enhanced Perls to label iron (Fey) deposits (Puccio, Simon et al. 2001).

Sirius red and fast green staining: Tissue sections were fixed with 10% paraformaldehyde in 0.1 M phosphate buffer (PBS), pH 7.4 for 10min and then ted with a saturated solution of picric acid containing 0.1% Direct red 80 (Sigma) for 2 min, washed with 0.5% l acetic acid solution followed by zed water, and subsequently incubated in 0.05% Fast Green solution for 5min, and then washed with 0.5% glacial acetic acid solution. Finally, sections were dehydrated in graded alcohols, cleared in Histosol Plus (Shandom) for 5 min and mounted using Pertex ng medium (Histolab ts AB). hanced Perls iron staining: Tissue sections were fixed with 10% paraformaldehyde in 0.1 M phosphate buffer (PBS), pH 7.4 for 20min and incubated in Perls solution (1%HCl,1% Potassium Ferrocyanide) for 30min. ng was enhanced by incubation in 0.025% 3’-3’-diaminobenzidine tetrahydrochloride (Sigma-Aldrich), 0.005% H202 in PBS buffer for 30 min, and then developed in the same buffer. Finally, ns were dehydrated in graded alcohols, cleared in ol Plus (Shandom) for 5 min and mounted using Pertex mounting medium (Histolab Products AB). _ 27 _ W0 2014/118346 Enzyme histochemical analyses: Succinate dehydrogenase (SDH) and Cytochrome C e (COX) activities were performed on 10um cryostat sections of tissues, as previously described o, Simon et al. 2001).

Electron microscopy analysis: Ultrathin sections (70 nm) of cardiac tissue were contrasted with uranyl acetate and lead citrate and examined with a Morgagni 268D electron microscope, as described previously (Puccio, Simon et al. 2001).

Immunofluorescence and image acguisition Cardiac and spinal cord tissue cryosections were fixed in 4% PFA for 10 min, washed and then permeabilized in methanol at -20°C for 20min. Sections were blocked and permeabilized at the same time with PBS, 1% NGS, 5% BSA, 0.3% Triton X-100 for 1h at room ature (RT) and then washed in PBS, 0.2% Tween 1% BSA 1% NGS (PBS-TBN).

Subsequently, tissues were incubated overnight (O/N) at 4°C with the rabbit polyclonal antibody against in (FXN935)(1/250) diluted in PBS-TBN (Puccio, Simon et al. 2001).

The Alexa fluor-5 94 goat abbit antibody (1/5 00) (Molecular ) was incubated for 2h at RT. Sections were stained with Hoechst and mounted using Aqua-Polymount mounting medium (Polysciences, Inc.). For co-immunolabelling of HA-tag and prohibitin, the tissue section were washed in PBS, 0.05% Tween and then blocked O/N at 4°C in M.O.M.TM Mouse Ig Blocking t (Vector Laboratories). Section were then incubated O/N at 4°C with the mouse monoclonal antibody to HA tag (1/150) (Covence) diluted in TM diluent (Vector Laboratories). After washing, sections were incubated for 1h at RT with the goat anti- mouse antibody conjugated to Alexa Fluor-594nm (1/500) (Molecular Probes) diluted in TM t. uently, ns were washed and blocked in PBS, 0.3% Triton, 2% NGS for 1h30 at RT, washed and incubated for 2h at RT with the rabbit polyclonal antibody to itin (1/150) (Abcam) diluted in PBS-BTN. The Alexa Fluor-488nm goat anti-rabbit antibody (1/500) (Molecular Probes) was ted 1h30 at RT with the goat anti- rabbit antibody conjugated to Alexa Fluor-488nm (Molecular Probes) diluted at 1/500 in PBS-BTN. Sections were stained with Hoechst and mounted using Aqua-Polymount mounting medium ciences, Inc).

Confocal analysis was performed on a Leica TCS SP2 upright confocal microsystem with a Plan Apo CS (numerical aperture 1.4) 63>< objective. Observation of whole cardiac cryosections was performed on a Leica Z16 APO A microsystem fitted with a QuanteM- S 12SC camera and combined with a 2x objective (39 mm working distance).

Quantitative real-time PCR Total Total RNA was extracted from frozen heart pulverized with the Precellys24 homogeniser (Bertin Technologies) and using TRI t (MRC) according to the manufacturer’s protocol and was treated with DNAse I treatment (Roche ences). cDNA was ted by e transcription using the Transcriptor Fist strand cDNA synthesis kit (Roche biosciences). Quantitative RT-PCR was performed using the SYBR Green I Master (Roche biosciences) and light Cycler 480 (Roche biosciences). 18S ribosomal RNA was used as al standard.

Enzyme activities Tissues were immediately frozen in liquid nitrogen. The activities of the respiratory chain enzyme SDH (complex II), the citric acid cycle enzymes isocitrate dehydrogenase, and mitochondrial and cytosolic aconitases were determined as described (Puccio, Simon et al. 2001).

Immunoblot is Extracts of tissues were frozen in liquid nitrogen, and then nized in lysis buffer containing Tris-HCl (280 mM, pH 6.8), 10% SDS, 50% glycerol. Total protein extract (10 µg or 50 µg) was analyzed on SDS-glycine polyacrylamide gels. Proteins were transferred to ellulose membranes blocked with 5% non-fat milk and then incubated with the different primary antibodies, polyclonal anti-frataxin (R1250 purified sera IGBMC, 1/1,000), anti-HA (Covance, 1/500), itochondrial aconitase (R2377 ed sera IGBMC, 1/20,000), anti-Ndufs3 (Invitrogen, 1/4,000), anti-SDH (Invitrogen, 1/4,000), anti- Rieske (Abcam, 1/5,000), anti-lipoic acid (Calbiochem, 1/5,000), anti-GAPDH (Millipore, 1/10,000) and monoclonal anti-beta-tubulin (2A2, IGBMC 0). Secondary antibody (goat anti-rabbit or anti-mouse IgG, respectively) coupled to peroxidase was diluted at 1/5,000 and used for detection of the reaction with Supersignal Substrate Western blotting (Pierce), according to the manufacturer’s instructions.

Statistical Analysis All data are ted as mean ± standard deviation of the mean (SD). Statistical analysis was carried out using Statview software (SAS Institute Inc). For statistical comparison of three experimental groups, one-way ANOVA followed by Scheffé’s post-hoc test was used.

A value of P<0.05 was considered significant. For tical comparison of two _ 29 _ experimental groups, the bilateral Student’s t-test was used. P <0.05 was considered significant.

Quantitative PCR on genomic DNA c DNA was extracted from heart by using a phenol-chloroform method.

CAG-FXN vector genome copy numbers were measured by quantitative PCR using the SYBR Green I Master (Roche Biosciences) and light Cycler 480 (Roche Biosciences). The vector genome copy number per cell (VGC) was evaluated as described (Piguet, Sondhi et al. 2012). The mouse genomic Adck3 sequence was used as internal control.

Results Three week-old MCK mice that do not exhibit yet any clinical, echocardiographic nor biochemical signs of c disease, received a single intravenous injection of AAVrth- CAG-hFXN at the dose of 5.4xlO13 vg/kg (n=9). Serial echocardiographic ements identified that the treatment efficiently prevented the development of the cardiac disease associated with fiataxin deficiency. While untreated MCK mice developed a rapidly progressing left ventricle hypertrophy associated with a massive geometric remodeling characterized by increased left-ventricular diastolic diameter, the treated MCK mice were inguishable from wild-type (WT) littermate animals (data not shown). In el, systolic filnction evaluated by the left-ventricular shortening fiaction (SF) and the cardiac output gradually decreased in untreated mice, while the treated MCK mice showed no sign of altered ventricular contractility (data not shown). The absence of echocardiographic phenotype in the treated MCK mice lead to normal growth (data not shown) and survival (35 weeks with no sign of disease), in contrast to untreated mice which die at 65:10 days (Fig. 1A). To assess the cellular and lar state ofthe cardiomyocytes, d MCK mice were sacrificed at 35 weeks of age i.e. more than triple lifespan of untreated mice. Consistent with the evolution towards heart failure, the expression of atrial natriuretic e (ANP) and the brain natriuretic e (BNP), two markers of pathology-induced stress m induced by hemodynamic overload was markedly increased in the heart from untreated mice at 8 weeks compared to WT (l9 and 7 times, respectively, 1) (Fig. 1B). In contrast, no difference could be detected in the expression level of these two markers between the treated MCK mice and the WT littermates, supporting the absence of pathology-induced stress programme due to hemodynamic overload (Fig. 1B). Furthermore, while the expression of sarcoplasmic reticulum Ca2+ ATPase (Serca2a), a critical determinant of cardiac relaxation sible for diastolic Ca2+ reuptake from cytosol was reduced in untreated mice (3.3 fold, p<0.01), treated MCK mice had normal Serca2a levels. Histological analysis confirmed a preserved overall heart organization in 35 week-old treated MCK mice, ed to the myocardial degeneration with cytoplasmic vacuolization in the necrotic myocytes observed in untreated mice at 8 weeks of age (data not shown). Furthermore, Sirius-red staining (data not shown) and en type I and III mRNA expression (data not shown) indicated the absence of myocardial post-necrotic is in treated s, in comparison to the massive interstitial fibrosis present in ted MCK mice at 8 weeks (data not shown).

Intravenous injection of AAVrh10-FXN led to robust viral transduction of the heart (20.85±6.3 vg/cell) and liver, but also of al muscle and dorsal root ganglia (data not shown). Western blot analysis using an anti-FXN antibody, which y detects human and mouse frataxins, demonstrated a significant overexpression (>10 fold) of AAVrh10- d frataxin compared to endogenous frataxin of WT mice (data not shown). Sustained expression of the AAVrh10-encoded frataxin was seen over 35 weeks (data not shown).

Mitochondrial import and maturation of frataxin was complete and non-saturated, as only the cleaved mature form of human frataxin was detected (data not shown).

Immunohistochemistry analysis using both anti-FXN and anti-HA antibodies showed a broad expression of human frataxin throughout the heart of the AAV treated MCK mice, with close to 100% of transduced cardiomyocytes in the LV, RV and septum, with some cardiomyocytes expressing higher levels (data not . Co-localization with prohibitin demonstrated the expected mitochondrial localization of human frataxin (data not shown).

In line with the ial function of frataxin in ting ar Fe-S cluster biogenesis, it is now commonly accepted that frataxin deficiency leads to a primary Fe-S cluster deficit followed by secondary mitochondrial iron accumulation. Indeed, while untreated MCK mice showed a strong deficit in the Fe-S ondrial aconitase (mAco) and succinate dehydrogenase (SDH) (41.3% and 79.8%, respectively) (data not shown), treated mice presented levels of activities similar to WT mates. Consistent with the widespread expression of hFXN in the heart after AAVrh10-CAG-hFXN injection, colorimetric staining of SDH activity confirmed the correction of Fe-S biogenesis in over 95% of cardiomyocytes _ 31 _ W0 2014/118346 (data not . While a substantial decrease in the levels of all analysed mitochondrial Fe-S proteins, was detected in untreated mice, as a result of the instability of the respective Fe-S apo-proteins, treated mutants had levels equivalent to WT (data not shown). Similarly, expression of human frataxin prevented the decrease in activity of the Fe-S enzyme lipoic acid synthase, indirectly demonstrated by normal levels of lipoic acid bound (x-ketoglutarate dehydrogenase (KGDH) and pyruvate dehydrogenase (PDH) in treated animals in comparison to untreated animals (data not . Consistent with the e of Fe-S cluster , no cellular iron accumulation was observed in the cardiac tissue oftreated mice (data not shown).

Furthermore, we did not detect any sign of cellular iron homeostasis perturbation in treated animals (data not shown). Finally, electron copy analysis demonstrated a normal sarcomere organization ofthe cardiomyocytes and mitochondria ultrastructure in treated mice.

Untreated animals showed sparse atrophied myof1brils and massive ondrial eration with abnormal collapsed or swollen cristae and iron accumulation (data not shown). All together, these data te that human frataxin gene transfer using AAVrth in pre-symptomatic MCK mice prevented the pment of the mitochondrial FRDA cardiomyopathy at the molecular, cellular and physiological level.

While preventing the onset of the cardiomyopathy is an important step, at a clinical point of view it s crucial to determine the therapeutic potential of this gene therapy approach when cardiac dysfunction is already t. Mutant MCK mice were intravenously injected with AAVrth-CAG-hFXN at the dose of 5.4x1013 vg/kg (n=9) at 7 weeks, when the ventricular remodeling and left ventricular ic dysfunction are established, with a major decrease in cardiac output (60::9% versus control values), attesting of cardiac failure. One week after injection at 8 weeks of age, the LV function was already significantly improved, with a 49::5% on fraction and a decrease in LV hypertrophy and dilation in the treated mutant mice, whereas ted animals presented typical signs of heart failure (Fig. 2A).

Echocardiographic parameters regarding cardiac fianction progressively improved to reach WT values at 11-12 weeks of age, demonstrating a te recovery of the ventricular systolic fianction and anatomy. The survival of the mice was significantly prolonged until at least 18 weeks of age (Fig. 2B). In accordance with the rapid reversion observed by echocardiography, human FXN was already strongly expressed one week after injection in heart of treated mutant mice and sustained over 22 weeks, with a mitochondrial localization (data not shown). Similarly, the ogy-induced stress program induced by hemodynamic ad, reflected by the expression ofANP and BNP, was significantly decreased one week _ 32 _ W0 2014/118346 after injection (8 weeks) in treated mice (Fig. 2C). By 22 weeks, the expression level of ANP and BNP of treated MCK mice was close to the expression level of WT animals, suggesting a normalization of the hemodynamic load. Furthermore, the sion of a progressively increased in treated mice between 8 and 22 weeks, indicating that diastolic Ca2+ transport was likely restored (Fig. 2C). The reversal and correction of the cardiac phenotype correlated with a progressive increased in Fe-S proteins activities, mAco and SDH, in levels of the Fe-S proteins Ndufs3, SDH, Rieske, as well as in the lipoic acid bound PDH and KGDH (Fig. 2D). At 22 weeks, some rare patches with low SDH activity were detected in the cardiac tissue of treated mice (data not , corresponding to fibrotic scar probably already present at the time of treated. Interestingly, collagen ng and sion (type I and III) showed that interstitial cardiac fibrosis stopped one week post injection (data not shown). Strikingly, a rapid correction of the ultrastructure of the cardiac muscle was also observed one week after injection, with normal sarcomere organization and with a massive decrease in mitochondria (data not shown). In correlation with a still lete recovery of the biochemical phenotype one week after treatment, the mitochondria in the treated animals showed some signs of ogy, with the ce of some swollen mitochondria presenting parallel stacks of cristae membranes (data not shown). However, by 22 weeks, sarcomeres and mitochondria organizations tely recovered with no sign of pathological change.

All together, these data indicate that AAVrth.CAG-hFXN treatment in symptomatic MCK mice resulted in a rapid clinical, echocardiographic and biochemical ement with a complete correction ofthe FRDA cardiomyopathy.

Conclusion Our data demonstrates that AAVrth-mediated transfer of hFXN gene in the myocardium of a mouse model of severe FRDA cardiomyopathy not only prevents the onset of the disease for a sustained period, but also can e heart failure and cardiac remodelling. The correction is extremely rapid and nt, with a striking reversal of the mitochondrial abnormalities and biochemical Fe-S ns deficit one week after ent.

Despite the ty of cardiac insufficiency at the time of treatment, the cardiac recovery is rapidly progressive, reaching normality within 4-5 weeks of treatment.

Indeed, the correction of mitochondrial ction in the mouse was associated with a progressive increase of sarcoplasmic reticulum Ca2+-ATPase (Serca2a) gene expression involved in sarcoplasmic reticulum calcium uptake from cytosol. Interestingly, decrease in the expression and activity of Serca2a has been identified in cardiomyocytes from failing human _ 33 _ W0 2014/118346 2014/051966 hearts. A rapid correction of the ultrastructure of the cardiac muscle was also observed and the interstitial cardiac fibrosis was stopped one week after treatment, preventing the dilation and massive remodelling of the cardiac tissue. Fibrosis is an early manifestation of FRDA cardiomyopathy and its importance in organ pathology and dysfilnction is relevant to a wide variety of diseases, including heart diseases.

In conclusion, delivery of a vector ng hFXN in a mammalian model of FRDA cardiomyopathy resulted in i) tion of the development of disease symptoms in asymptomatic individuals and ii) al of disease symptoms in individuals who already exhibited cardiomyopathy, biochemical Fe-S cluster impairment, mitochondrial dysfilnction and titial cardiac fibrosis.

Thus, the use of a gene that can restore energy failure may be useful for the treatment and the prevention of a cardiomyopathy due to energy failure (like the use of FXN gene in the case of cardiomyopathy associated with Friedreich ataxia as explained in the examples).

Example 2: Determination of the minimal dose of AAVrth.CAG-FXN vector injected inducing a detectable level of cardiac transgene expression al & Methods The dose-response evaluation was ted in 5 weeks-old MCK mice with early- stage systolic dysfunction using the same experimental design as described above. All animal procedures and experiments were approved by the local ethical tee for Animal Care and Use th 2012-016). Briefly, mice were anesthetized and then injected retro-orbitaly with lOOuL of AAVrth.CAG-FXN vector solution diluted in saline solution, for a final injected-dose of 5x10", 2.5x1013, 1x1013 and 5x1012 vg/kg, respectively (Table 1). Age matched WT mice were injected with lOOuL of saline solution, as control. Survival, growth and cardiac fianction were ted weekly up to 12 weeks of age (Fig. 1). All mice were sacrificed at 12 weeks of age. Heart was sampled and dissected transversally in order to snap freeze a small transversal section of l-2mm thick from the middle of the left-ventricle and to process separately the apex and the middle-to-base of the heart which were embedded in OCT Tissue Tek and snap-frozen in isopentane chilled in liquid nitrogen. The OCT-included heart s were cut alternatively at 5 and lOOum from the apex to the base of the heart. H&E staining as well as SDH and COX activities were performed on Sum heart sections, at the level of the apex, middle and base of the left ventricle. g of whole heart sections was performed at high magnification with the Hamamatsu omer 2.0 slide scanner and analysis were performed using ImageJ following the color threshold method. RNA and DNA were extracted from 100µm thick cryosection following the trizol-based protocol as described above. The apex, middle and base of the left-ventricle were analyzed separately. Vector copy per diploid genome and relative expression of transgene were quantified as described above.

Results The dose of 5x1013 vg/kg used in the original study allows transduction of near 100% of the cardiomyocytes, a transduction efficiency that will very ly be achieved in humans.

Therefore, to determine the lower therapeutic threshold that is ed to rescue the cardiomyopathy associated with frataxin deficiency in the MCK mice, a dose-response study was performed. MCK mice with early stage systolic dysfunction were intravenously injected with AAVrh10-CAG-hFXN at a dose of 5x10¹³, 2.5x10¹³, 1x10¹³, and 5x1012 vg/kg (n=2-3 per dose), respectively, at 5-weeks of age. Serial rdiographic measurements were performed weekly up to 12 weeks of age. Although some variability was observed, MCK mice injected at the highest doses (5x10¹³, ¹³ and 1x10¹³ vg/kg) did not show any hemodynamic nor morphological y up to 12 weeks of age (Fig. 3a-d, Table 1), demonstrating efficient correction of the early stage ic dysfunction and tion of cardiac e onset associated with FXN deficiency. At the lower dose, 5x1012 vg/kg, while the hemodynamic and logical measurements are stable and consistent with a correction of the early stage systolic dysfunction and prevention of cardiac failure onset up to 10 weeks of age, a systolic dysfunction (Fig. 1 a-b) accompanied by a left ventricular remodeling was observed starting 10 weeks of age leading to a rapid declined of the cardiac output.

All animals in each dose tested were sacrificed at 12 weeks of age to determine the transduction efficiency by measuring the vector genome copy per diploid genome and the percentage of corrected cardiomyocytes by SDH staining (Table 1). While variability was observed in the number of vector genome copy per cell, in particular at lower doses, we find a correlation n the vector genome copy, the percentage of SDH ve cells (which correlates very well with the percentage of frataxin expressing cells) and cardiac function (Table 1). Together, these results suggest that at 5 weeks of age, the therapeutic threshold appears to be around 2-3 l, with 50-60% of SDH positive cardiomyocytes.

Example 3: ent transduction of monkey and pig cardiomyocytes after intramyocardial injection of AAVrh10 vector _ 35 _ W0 2014/118346 Material & Methods AAVrth-CAG-GFP vector was administered using an identical cardiac surgery procedure in monkey and pig. ing general anesthesia, a median stemotomy was med. The AAVrth-CAG-GFP vector was stered directly to the myocardium of the left cle as subepicardial injections. In monkey, 9 cardial injections were performed, with 3 subepicardial injections 0.5 cm apart in 3 spots (l.66xlOIO vg of vector per injection, 40 ul/injection). The total dose of injected vector per spot was 5x1010 vg. In pig, l8 subepicardial injections were performed with 3 subepicardial injections in 6 different spots (3 vector injections per spot). In one spot, 3 vector injections were made 0.5 cm apart (as in monkey) and 1.66 x1010 vg (as in monkey) of vector diluted in 40 ul of PBS was ed at each site. In the 5 other spots, vector injection was made 1 cm apart with 10 vg/injection in 3 spots and 1.25x1011 vg/injection in 2 spots. The total dose of injected vector per spot was therefore 5x1010 vg (1 spot), 2.5xlO11 vg (3 spots) and 3.75xlO11 vg (2 spots).

The cardiac surgery procedure and vector injection were associated with no adverse event, even minimal. In pig, cardiac echography performed 10 days afier vector injection was normal, showing no s in respect to cardiac parameters observed immediately prior to cardiac surgery.

Monkey and pig were euthanized 3 weeks afier vector injection. The lefi ventricle was dissected from the ing part of the heart and trimmed in 4 (monkey) or 10 (pig) separate pieces along antero-longitudinal axis. Each piece of lefi ventricle was then cut in 20 mm-thick slices and one out of two slices were processed for directed visualization of GFP expression under fluorescence microscopy. The other slices were processed for DNA extraction to measure vector copy number. Percentage of GFP-positive cardiomyocytes was assessed using an in-house software. Vector copy number was assessed using quantitative PCR with primers ing the ITR2 of AAVrth-CAG-GFP vector and the endogenous albumin gene. Vector copy number was assessed using the cycle (Ct) threshold to detect albumin or ITR2 ces. At autopsy, there was no macroscopic lesion of lefi ventricle in monkey and pig.

In monkey, there was a mild inflammatory reaction in the myocardium around 2 injection sites in which GFP expression was observed. There was no inflammatory reaction in the dium ofvector-injected pig.

Results _ 36 _ W0 18346 2014/051966 Intravenous injection of AAVrth-CAG-hFXN vector will likely not be feasible in FRDA patients because it would require the injection of a very high dose of vector that could result in significant adverse events, mostly related to an immune reaction against the AAVrh10 capsid in the periphery. Direct intra-myocardial injection of gene therapy vector through subepicardial injections following mini-thoracotomy has proven to be safe over an average of nearly 12-year follow-up period (Rosengart TK et al, Hum Gene Ther, 2 :203-208, 2013). The transduction cy ofAAV of a given serotype in a given tissue can be species specific. At last efficacy of hFXN gene transfer in cardiomyocytes must be evaluated in a large animal whose heart size is r to human. To answer to these important pre-clinical steps towards a gene therapy trial in ts with FRDA cardiomyopathy, AAVrh10 vector expressing the reporter green fluorescent protein (GFP) under the control of CAG promoter was injected directly into the myocardium of one monkey (Macaca fascicularis) and one pig.

Monkey is the best animal model to anticipate that the observed transduction efficacy of an AAV vector of a given serotype in a given tissue will be similar in human patients. Two-three year-old pigs of 20-30 kg have a size of the heart, in particular of the left ventricle, which is r to .

AAVrth-CAG-GFP vector was ly delivered in the myocardium of a monkey through epicardial injections. Three separate spots of myocardium were injected with 3 vector injections per spot. At a non-optimized dose of 5x1010 vector genome (vg) per spot, GFP sion was observed in 60% of cardiomyocytes (data not shown). The expression of GFP was similar in the 3 spots. The mean volume in each spot in which GFP was expressed was 200 m3. The mean vector copy number/cell was 1.2.

AAVrth-CAG-GFP vector was then directly delivered in the myocardium of a pig through epicardial ions. Six separate spots of myocardium were injected with 3 vector injections per spot. The spots were injected with different doses of vector : 5x1010 vg (spot#1), 2.5x1011 vg (spots# 2, 3 and 4) and 3.75x1011 vg (spots# 5 and 6). GFP expression was observed in 10% of cardiomyocytes in spot#1 whereas GFP expression was observed in 50 to 95% of cardiomyocytes in 2, 3 and 4. Injection of higher dose of AAVrh10- CAG-GFP vector in 5 and 6 did not result in significant better transduction of cardiomyocytes. The mean volume in spots#2 to 6 in which GFP was expressed was 330 mm3. The mean vector copy number/cell in spots #2 to 4 was 1.8.

Altogether, these results demonstrate that cardiomyocytes of monkey and pig are very well transduced by 0 vector following the direct administration of the vector to the myocardium. Results obtained in monkey indicates that 30 injections of higher dose of _ 37 _ W0 2014/118346 AAVrh10 vector should be sufficient to transduce > 50% of myocytes of the left ventricle in human. Thirty epicardial viral vector injections following mini-thoracotomy has proven to be feasible (l-hour surgical procedure) and safe in humans (Rosengart TK et al, Hum Gene Ther, 2 :203-208, 2013). Results of transduction efficacy with 0 in pig cardiomyocytes indicates that this porcine animal model will be suitable to evaluate the total dose of AAVrth-CAG-hFXN vector that it will be needed to inject in the left cle myocardium ofFRDA patients to express frataxin in > 50% of their cardiomyocytes.

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Claims (6)

1. Use of an adeno-associated virus rh10 (AAVrh10) vector in the preparation of a medicament for preventing or treating myopathy associated with Friedreich 5 ataxia in a human, wherein the AAVrh10 vector comprises a nucleic acid ce of a frataxin (FXN)-encoding nucleic acid or a fragment thereof, and n the AAVrh10 vector is formulated for administration to the subject at a dose of about 5 × 1012 viral genomes (vg)/kg.

2. The use according to claim 1, wherein said FXN encoding nucleic acid encodes for 10 the amino acid sequence SEQ ID NO:2.

3. The use according to according to claim 1 or 2, wherein the vector comprises the nucleic acid sequence SEQ ID NO:1.

4. The use according to any one of claims 1 to 3 wherein the vector is formulated for administration by intravenous ion. 15

5. A ceutical composition for preventing or treating cardiomyopathy associated with Friedreich ataxia in a human in need thereof, comprising an ssociated virus rh10 10) vector comprising a nucleic acid encoding a frataxin (FXN) or a fragment thereof and a pharmaceutically acceptable vehicle, wherein the AAVrh10 vector is formulated for administration to the subject at a 20 dose of about 5 × 1012 viral genomes (vg)/kg.

6. The use according to claim 1, substantially as herein described with reference to any one of the Examples and/or