U.S. patent application number 17/103783 was filed with the patent office on 2021-11-04 for methods and pharmaceutical composition for the treatment and the prevention of cardiomyopathy due to energy failure.
The applicant listed for this patent is ASSISTANCE PUBLIQUE - HOPITAUX DE PARIS (APHP), CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE (CNRS), CORNELL UNIVERSITY, INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE (INSERM), UNIVERSITE DE STRASBOURG, UNIVERSITE PARIS-SUD XI. Invention is credited to Patrick AUBOURG, Pierre BOUGNERES, Ronald G. CRYSTAL, Helene Monique PUCCIO.
Application Number | 20210340565 17/103783 |
Document ID | / |
Family ID | 1000005724974 |
Filed Date | 2021-11-04 |
United States Patent
Application |
20210340565 |
Kind Code |
A1 |
PUCCIO; Helene Monique ; et
al. |
November 4, 2021 |
METHODS AND PHARMACEUTICAL COMPOSITION FOR THE TREATMENT AND THE
PREVENTION OF CARDIOMYOPATHY DUE TO ENERGY FAILURE
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.
Inventors: |
PUCCIO; Helene Monique;
(Illkirch, FR) ; AUBOURG; Patrick; (Le Kremlin-Bic
tre, FR) ; CRYSTAL; Ronald G.; (New York, NY)
; BOUGNERES; Pierre; (Le Kremlin-Bic tre, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE
(INSERM)
CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE (CNRS)
UNIVERSITE DE STRASBOURG
CORNELL UNIVERSITY
UNIVERSITE PARIS-SUD XI
ASSISTANCE PUBLIQUE - HOPITAUX DE PARIS (APHP) |
Paris
Paris
Strasbourg
Ithaca
Orsay Cedex
Paris |
NY |
FR
FR
FR
US
FR
FR |
|
|
Family ID: |
1000005724974 |
Appl. No.: |
17/103783 |
Filed: |
November 24, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15916907 |
Mar 9, 2018 |
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17103783 |
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14764065 |
Jul 28, 2015 |
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PCT/EP2014/051966 |
Jan 31, 2014 |
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15916907 |
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13756651 |
Feb 1, 2013 |
9066966 |
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14764065 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2750/14071
20130101; A61K 9/0019 20130101; C12N 2750/14132 20130101; C12N 7/00
20130101; C12N 2750/14032 20130101; C12N 2750/14143 20130101; A61K
48/0016 20130101; C12N 15/86 20130101; C12Y 116/03001 20130101;
C07K 14/47 20130101; C12N 2750/14171 20130101; A61K 38/44 20130101;
A61K 48/005 20130101; A61K 31/7088 20130101; A61K 48/00
20130101 |
International
Class: |
C12N 15/86 20060101
C12N015/86; A61K 31/7088 20060101 A61K031/7088; A61K 38/44 20060101
A61K038/44; C12N 7/00 20060101 C12N007/00; A61K 9/00 20060101
A61K009/00; A61K 48/00 20060101 A61K048/00; C07K 14/47 20060101
C07K014/47 |
Claims
1. 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.
2. The method according to claim 1, wherein the cardiomyopathy due
to energy failure is a cardiomyopathy associated with Friedreich
ataxia and the gene that can restore energy failure is the frataxin
(FXN) encoding nucleic acid.
3. The method according to claim 1, wherein said FXN encoding
nucleic acid encodes for the amino acid sequence SEQ ID NO:2.
4. The method according to claim 1, wherein the vector comprises
the nucleic acid sequence SEQ ID NO:1.
5. The method according to claim 1, wherein the vector is selected
from the group consisting of adenovirus, retrovirus, herpesvirus
and Adeno-Associated Virus (AAV) vectors.
6. The method according to claim 5, wherein the vector is an AAV
vector.
7. The method according to claim 6, wherein the AAV vector is an
AAV1, AAV2, AAV3, AAV4, AAS, AAV6, AAV7, AAV8, AAV9, AAVrh10 vector
or any AAV derived vector.
8. The method according to claim 7, wherein the AA V vector is an
AA Vrh10 vector.
9. The method according to claim 1, wherein the vector is
administered intracoronary or directly into the myocardium of the
subject.
10. The method according to claim 1, wherein the vector is
administered by intravenous injection.
11. (canceled)
12. (canceled)
13. A method for reversing symptoms of 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.
14. 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.
15. A vector according to claim 14, wherein the vector comprises a
frataxin (FXN) encoding nucleic acid.
16. A vector according to claim 14, wherein the cardiomyopathy is a
cardiomyopathy associated with Friedreich ataxia.
17. A vector according to claim 14, wherein the vector is an
AAVrh10 vector.
18. (canceled)
19. (canceled)
20. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/764,065, which adopts the international
filing date of Jan. 31, 2014, which is a National Phase application
under 35 U.S.C. .sctn. 371 of International Application No.
PCT/EP2014/051966 filed Jan. 31, 2014, which claims benefit of
priority to U.S. patent application Ser. No. 13/756,651, filed Feb.
1, 2013, now U.S. Pat. No. 9,066,966, each of which is incorporated
herein by reference in its entirety.
SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
[0002] The content of the following submission on ASCII text file
is incorporated herein by reference in its entirety: a computer
readable form (CRF) of the Sequence Listing (file name:
627002000102seqlist.txt, date recorded: Mar. 8, 2018, size: 12
KB).
FIELD OF THE INVENTION
[0003] 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.
[0004] 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.
BACKGROUND OF THE INVENTION
[0005] Friedreich ataxia (FRDA), an autosomal progressive recessive
neurodegenerative disorder associated with cardiomyopathy, is
caused by reduced expression of the mitochondrial protein, frataxin
[V. Campuzano et al., 1996 and V. Campuzano et al., 1997]. The
cardiomopathy associated with FRDA is hypertrophic. As the disease
progresses, there is a natural transition from hypertrophy to
dilation, with death of cardiomyocytes replaced by fibrotic tissue
leading to systolic and diastolic dysfunction [R. M. Payne et al.,
2012]. Impaired myocardial perfusion reserve index (MPRI)
associated with microvascular dysfunction and fibrosis occurs even
prior to the onset of overt cardiomyopathy. Consistent with
impaired mitochondrial respiratory chain function that leads to
energy deficit, phosphorus magnetic resonance spectroscopy shows
reduced ATP production in patient heart, which strongly correlates
with the degree of cardiac hypertrophy. Cardiac dysfunction,
predisposing to congestive heart failure and supraventricular
arrhythmias, is the primary mode of death in .about.60% of patients
with FRDA.
[0006] Although the function of frataxin is still under
investigation, available evidences support a role as an activator
of iron-sulfur (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 cysteine desulfurase
activity [Colin et al., 2013].
[0007] Fe--S clusters are essential prosthetic groups for many
proteins with a variety of functions and subcellular localizations.
Frataxin deficiency leads to impairment of Fe--S cluster-containing
proteins, altered cellular iron metabolism, mitochondrial
dysfunction 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]. Furthermore, the MCK mouse
demonstrated that hypertrophy and mitochondrial Fe--S cluster
protein defects precede mitochondrial iron accumulation and
increase sensitivity to oxidative stress.
[0008] 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
[0009] In the present invention, the therapeutic effect of an
AVVrh10 vector carrying a human frataxin cDNA on the cardiac
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 disease
symptoms in asymptomatic individuals and ii) reversal of disease
symptoms in individuals who already exhibited cardiomyopathy,
mitochondrial dysfunction and the biochemical impairment associated
with frataxin deficiency. Of particular note is that the inventors
showed that delivery 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 cardiac function.
[0010] More generally, the inventors show that by restoring an
essential mitochondrial function with the use of the
nuclear-encoded FXN gene, and thereby increasing the energy
production of the mitochondria, the myocardium function can be
restored and the interstitial cardiac fibrosis stopped. Considering
that inefficient energy utilization and increased energy demand by
the sarcomere have been suggested as a key consequence of many, if
not all, hypertrophic cardiomyopathy associated mutations (Sweeney
H L et al., 1998), they results demonstrate that the use of a gene
that can restore energy failure may be useful for preventing or
treating cardiomyopathy linked to energy failure.
[0011] Thus, the 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.
[0012] 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.
DETAILED DESCRIPTION OF THE INVENTION
Methods of the Invention
[0013] A first object of the 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.
[0014] As used herein the term "cardiomyopathy due to energy
failure" denotes a deterioration of the function of the myocardium
leading to heart failure, cardiac remodelling, long-term high blood
pressure, heart valve problems, impaired calcium cycling
sensitivity, disturbed biochemical stress sensing, altered energy
homeostasis due but not limited to mitochondrial dysfunction and
fibrosis.
[0015] 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.
[0016] 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 primary carnitine
deficiency, LCHAD, translocase, VLCAD.
[0017] In another particular embodiment, the cardiomyopathy due to
energy failure may be a cardiomyopathy associated with Friedreich
ataxia.
[0018] 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 dysfunction.
[0019] In a particular embodiment, the gene that can restore energy
failure may be a nuclear gene encoding a subunit of pyruvate
dehydrogenase complex, a nuclear or a mitochondrial gene coding for
a subunit of Complex I, III, IV or V involved in the oxidative
phosphorylation; a mitochondrial gene encoding transfer RNA, a gene
involved in the biogenesis of mitochondria such as SIRT1, a gene
involved in the fusion of mitochondria such as OPA1, a gene
involved in the fission of mitochondria such as FIS1 or a gene
involved in the oxidation of fatty acid such as the very long-chain
specific acyl-CoA dehydrogenase.
[0020] In a particular embodiment, the gene that can restore energy
failure is the frataxin (FXN) gene.
[0021] 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
diagnosed as having it or which does not have any clinical
symptoms.
[0022] 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 disorder 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 amount" to a patient is such
an amount which induces, ameliorates, stabilises, slows down the
progression or otherwise causes an improvement in the pathological
symptoms, disease progression or physiological conditions
associated with or resistance to succumbing to a disorder.
[0023] 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,
preferably a human, with a cardiomyopathy due to energy failure and
more particularly a subject with a cardiomyopathy associated with
Friedreich ataxia. Subject with a cardiomyopathy associated with
Friedreich ataxia presents some cardiac symptoms which may be, but
are not limited to, a decrease of ejection fraction, increase of
ventricular mass or cardiac hypertrophy. Thus, the method of the
invention will be very useful to treat subject with such disease
(Friedreich ataxia) presenting such symptoms.
[0024] 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.
[0025] 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 vivo when placed under the control of appropriate
regulatory sequences. The boundaries 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
eukaryotic DNA, and even synthetic DNA sequences.
[0026] In a particular embodiment, 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.
[0027] In a particular embodiment, the invention relates to a
method for 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.
[0028] In a particular embodiment, the invention relates to a
method for reversing or stabilizing symptoms of 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.
[0029] 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.
[0030] In a particular embodiment, the invention relates to a
method for reversing the dysfunction of cardiac mitochondria
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.
[0031] In a particular embodiment, the invention relates to a
method for improving the cardiac mitochondria 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.
[0032] In a particular embodiment, the invention relates to a
method for restoring cardiac function 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) encoding nucleic
acid.
[0033] In a particular embodiment, the invention relates to a
method for improving cardiac function in a subject suffering of a
cardiomyopathy associated with Friedreich ataxia comprising
administering to said subject a therapeutically effective amount of
a vector which comprises a frataxin (FXN) encoding nucleic
acid.
[0034] 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.
[0035] In another particular embodiment, the invention relates to a
method for treating a cardiomyopathy associated with Friedreich
ataxia in a 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.
[0036] As used herein, the terms "asymptomatic" or
"pre-symptomatic" denotes a subject with the disease (Friedreich
ataxia) as defined by a genetic diagnosis (see for review Lynch D R
et al., 2002) but with no clinical cardiac symptom.
[0037] As used herein, the terms symptomatic denotes a subject with
the disease (Friedreich ataxia) as defined by a genetic diagnosis
and with the presence of cardiac symptoms (cardiac hypertrophy,
fibrosis, decreased myocardiac perfusion reserve index, impaired
cardiac or skeletal muscle mitochondrial respiratory chain
function, subclinical cardiomyopathy, supraventricular arrhythmias,
heart failure, systolic left ventricular dysfunction, fatigue . . .
).
[0038] The FXN gene encodes the protein frataxin. This frataxin is
a protein localized to the mitochondrion. The frataxin is involved
in assembly of iron-sulfur clusters by regulating iron entry and
the activity of the cysteine desulfurase. 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 NO:2.
TABLE-US-00001 The sequence of the nucleic acid of the frataxin
(cDNA) is (SEQ ID NO: 1): agtctccctt gggtcagggg tcctggttgc
actccgtgct ttgcacaaag caggctctcc atttttgtta aatgcacgaa tagtgctaag
ctgggaagtt cttcctgagg tctaacctct agctgctccc ccacagaaga gtgcctgcgg
ccagtggcca ccaggggtcg ccgcagcacc cagcgctgga gggcggagcg ggcggcagac
ccggagcagc atgtggactc tcgggcgccg cgcagtagcc ggcctcctgg cgtcacccag
cccagcccag gcccagaccc tcacccgggt cccgcggccg gcagagttgg ccccactctg
cggccgccgt ggcctgcgca ccgacatcga 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 tgtactccca
cgacggcgtg tccctccatg agctgctggc cgcagagctc actaaagcct taaaaaccaa
actggacttg tcttccttgg cctattccgg aaaagatgct tgatgcccag ccccgtttta
aggacattaa aagctatcag gccaagaccc cagcttcatt atgcagctga ggtctgtttt
ttgttgttgt tgttgtttat tttttttatt cctgcttttg aggacagttg ggctatgtgt
cacagctctg tagaaagaat gtgttgcctc ctaccttgcc cccaagttct gatttttaat
ttctatggaa gattttttgg attgtcggat ttcctccctc acatgatacc ccttatcttt
tataatgtct tatgcctata cctgaatata acaaccttta aaaaagcaaa ataataagaa
ggaaaaattc caggagggaa aatgaattgt cttcactctt cattctttga aggatttact
gcaagaagta catgaagagc agctggtcaa cctgctcact gttctatctc caaatgagac
acattaaagg gtagcctaca aatgttttca ggcttctttc aaagtgtaag cacttctgag
ctctttagca ttgaagtgtc gaaagcaact cacacgggaa gatcatttct tatttgtgct
ctgtgactgc caaggtgtgg cctgcactgg gttgtccagg gagacctagt gctgtttctc
ccacatattc acatacgtgt ctgtgtgtat atatattttt tcaatttaaa ggttagtatg
gaatcagctg ctacaagaat gcaaaaaat ttccaaagac aagaaaagag gaaaaaaagc
cgttttcatg agctgagtga tgtagcgtaa caaacaaaat catggagctg aggaggtgcc
ttgtaaacat gaaggggcag ataaaggaag gagatactca tgttgataaa gagagccctg
gtcctagaca tagttcagcc acaaagtagt tgtccctttg tggacaagtt tcccaaattc
cctggacctc tgcttcccca tctgttaaat gagagaatag agtatggttg attcccagca
ttcagtggtc ctgtcaagca acctaacag ctagttctaa ttccctattg ggtagatgag
gggatgacaa agaacagttt ttaagctata taggaaacat tgttattggt gttgccctat
cgtgatttca gttgaattca tgtgaaaata atagccatcc ttggcctggc gcggtggctc
acacctgtaa tcccagcact tttggaggcc aaggtgggtg 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
tcctttattg tacccttact gggttaatcg tattatacca cattacctca attaatat
tactgacctg cactttatac aaagcaacaa gcctccagga cattaaaatt catgcaaagt
tatgctcatg ttatattatt ttcttactta aagaaggatt tattagtggc 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 tgggacacag cacaaaagag
gtatgcagtg gggctgctct gacatgaaag tggaagttaa ggaatctggg ctcttatggg
gtccttgtgg gccagccctt caggcctatt ttactttcat tttacatata gctctaattg
gtttgattat ctcgttccca aggcagtggg agatccccat ttaaggaaag aaaaggggcc
tggcacagtg gctcatgcct gtaatcccag cactttggga ggctgaggca agtgtatcac
ctgaggtcag gagttcaaga ccagcctggc caacatggca aaatcccgtc tctactaaaa
atattaaaaa attggctggg cgtggtggtt cgtgcctata atttcagcta ctcaggaggc
tgaggcagga gaatcgctgt aacctggggg gtggaggttg cagtgagacg agatcatgcc
acttcactcc agcctggcca acagagcca actccgtctc aaataaataa ataaataaat
aaagggactt caaacacatg aacagcagcc aggggaagaa tcaaaatcat attctgtcaa
gcaaactgga aaagtaccac tgtgtgtacc aatagcctcc ccaccacaga ccctgggagc
atcgcctcat ttatggtgtg gtccagtcat ccatgtgaag gatgagtttc caggaaaagg
ttattaaata ttcactgtaa catactggag gaggtgagga attgcataat acaatcttag
aaaacttttt tttccccttt ctattttttg agacaggatc tcactttggc actcaggctg
gaggacagtg gtacaatcaa agctcatggc agcctcgacc tccctgggct tgggcaatcc
tcccacaggt gtgcacctcc atagctggct aatttgtgta ttttttgtag agatggggtt
tcaccatgtt gcccaggctg gtctctaaca cttaggctca agtgatccac ctgcctcgtc
ctcccaagat gctgggatta caggtgtgtg ccacaggtgt tcatcagaaa gctttttcta
ttatttttac cttcttgagt gggtagaacc tcagccacat agaaaataaa atgttctggc
atgacttatt tagctctctg gaattacaaa gaaggaatga ggtgtgtaaa agagaacctg
ggtttttgaa tcacaaattt agaatttaat cgaaactctg cctcttactt gtttgtagac
actgacagtg gcctcatgtt tttttttttt ttaatctata aaatggagat atctaacatg
ttgagcctgg gcccacaggc aaagcacaat cctgatgtga gaagtactca gttcatgaca
actgttgttc tcacatgcat agcataattt catattcaca ttggaggact tctcccaaaa
tatggatgac gttccctact caaccttgaa cttaatcaaa atactcagtt tacttaactt
cgtattagat tctgattccc tggaaccatt tatcgtgtgc cttaccatgc ttatatttta
cttgatcttt tgcatacctt ctaaaactat tttagccaat ttaaaatttg acagtttgca
ttaaattata ggtttacaat atgctttatc cagctatacc tgccccaaat tctgacagat
gcttttgcca cctctaaagg aagacccatg ttcatagtga tggagtttgt gtggactaac
catgcaaggt tgccaaggaa aaatcgcttt acgcttccaa ggtacacact aagatgaaag
taattttagt ccgtgtccag ttggattctt ggcacatagt tatcttctgc tagaacaaac
taaaacagct acatgccagc 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 gggattacag gcgtgagcca ctgcacccag ccagaaatgc cttctaatct
ttggtttatc ttaattagcc aggacacttg gagtgcatcc 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 aagaagggcc tctgctgtcc acttgtgttt
ctgtgatctg tgggaacatt gttaacgcca catcttgacc tcaaattgtt tagctcctgg
ccagacacgg tggctcacac ctgtaatccc agcactttga gaggctgagg caggtggatc
acctgaggtt aggagttcga ggccagcctg gtcaacatgg taaaaccccg cctctactaa
aaatacaaaa attagctggc cgtagtggcg cacgcctgtt atcccagcta ctcgggaggc
tgaggcagga gaattgcttg aacctgggtg gtggaggttg cagtgagccg agattacacc
actgcactcc agcctgggtg acaagaggga aactccatta aaaaaatgta attcccgtgt
ctgccatctt aagtgtaaag gtggctaaat tatatagaaa aataagacaa tatcatttcc
caattacatt cctttcctac cgcactctat gatgctagct gagatttttc caaaagaaaa
tggcttaaat aaaacccta gagaaagaaa aactttaaat ccctccaaag ctcaaaagta
atagaaacag atgagtttgg agtcaggatt tctctgtaag attgcctagg ctgtgtactg
cacatctcca ggtgccactg ttgacagaga ttataactac aatgtgaagt gaatggtgcc
actgacagtt atgcaaaccg tccagagcat agccacctga tcctgctggg attcctcttg
ccagtccatc agcagttccc cttgaaagtt tcaccaaaca tcccttaaat ctgccctctc
ctgcccgtcc ccagtggagg tcctcatcat ttttcacctg catttttgca ggagctttct
tatatccacc ttcctccttt tctctcagcc catcatctag ctacacagtc tccagggtaa
gctttcagaa aggcaatctc ttgtctgtaa aacctaagca ggaccaaggc caagtttctt
agcctgaaaa atgtgctttt ctgactgaac tgttcaggca ctgactctac atataattat
gcttttctac cccctcacac tcaacacttt gactccagca atcccaaatc cccagatccc
taagtgtgct gtgctatttt cacgtggctc tcagacttgg ccagtgctgt 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):
MWTLGRRAVAGLLASPSPAQAQTLTRVPRPAELAPLCGRRGLRTDIDA
TCTPRRASSNQRGLNQIWNVKKQSVYLMNLRKSGTLGHPGSLDETTYE
RLAEETLDSLAEFFEDLADKPYTFEDYDVSFGSGVLTVKLGGDLGTYV
INKQTPNKQIWLSSPSSGPKRYDWTGKNWVYSHDGVSLHELLAAELTK
ALKTKLDLSSLAYSGKDA.
[0039] In a particular embodiment, the invention provides a nucleic
acid construct comprising sequence SEQ ID NO:1 or a variant thereof
for treating cardiomyopathy associated with Friedreich ataxia.
[0040] 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_001161706 and
NM_181425), etc. The term variant also includes FXN gene sequences
from other sources or organisms. Variants are preferably
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 nucleic acid sequences,
which hybridize to a sequence as defined above (or a complementary
strand thereof) under stringent hybridization conditions. Typical
stringent hybridisation conditions include temperatures above
30.degree. C., preferably above 35.degree. C., more preferably in
excess of 42.degree. 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.
[0041] In a particular embodiment, the FXN-encoding nucleic acid is
a fragment of the SEQ ID NO:1 which encodes for the amino acid
sequence 81-210 of the SEQ ID NO:2 (named variant "81-210") or a
variant thereof, "variant" having the meaning provided above with
respect to nucleotide sequence identity and hybridization.
[0042] In a another particular embodiment, a sequence known as
mitochondrion-targeting signal or mitochondrial targeting signal
may be added to the FXN-encoding sequence or variant thereof,
including, for example 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.
[0043] A MTS sequence can be identified within a protein or nucleic
acid sequence by a person of ordinary skill in the art.
[0044] Most mitochondrion-targeting peptides consist of a
N-terminal pre-sequence of about 15 to 100 residues, preferably of
about 20 to 80 residues. They are enriched in arginine, 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 amphiphilic alpha-helix.
[0045] A complete description of a method to identify a MTS is
available in: M. G. 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.
[0046] In another embodiment, the invention relates to a method for
use in the prevention or treatment of diseases 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.
[0047] In another embodiment, the invention relates 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 subject a therapeutically effective amount of
a vector which comprises a nucleic acid sequence of a gene that can
restore energy failure.
[0048] In another particular embodiment, the invention relates 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 subject a therapeutically
effective amount of a vector which comprises a frataxin (FXN)
encoding nucleic acid.
[0049] 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.
[0050] In a particular embodiment, the invention relates to a
vector which comprises a frataxin (FXN) encoding nucleic acid for
use in the treatment or prevention of a cardiomyopathy associated
with Friedreich ataxia in a subject in need thereof.
[0051] In a particular embodiment, 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.
[0052] In a particular embodiment, the invention relates to a
vector which comprises a frataxin (FXN) encoding nucleic acid for
reversing or stabilizing symptoms of cardiomyopathy associated with
Friedreich ataxia in a subject in need thereof.
[0053] In a particular embodiment, the invention relates to a
vector which comprises a frataxin (FXN) encoding nucleic acid for
reversing the dysfunction of cardiac mitochondria associated with
Friedreich ataxia in a subject in need thereof.
[0054] In a particular embodiment, the invention relates to a
vector which comprises a frataxin (FXN) encoding nucleic acid for
improving the cardiac mitochondria associated with Friedreich
ataxia in a subject in need thereof.
[0055] In a particular embodiment, the invention relates to a
vector which comprises a frataxin (FXN) encoding nucleic acid for
restoring cardiac function in a subject suffering of a
cardiomyopathy associated with Friedreich ataxia.
[0056] In a particular embodiment, the invention relates to a
vector which comprises a frataxin (FXN) encoding nucleic acid for
improving cardiac function in a subject suffering of a
cardiomyopathy associated with Friedreich ataxia.
[0057] In a particular embodiment, the invention relates to a
vector which comprises a frataxin (FXN) encoding nucleic acid for
use in the treatment a cardiomyopathy associated with Friedreich
ataxia in an asymptomatic or pre-symptomatic subject in need
thereof.
[0058] In a particular embodiment, the invention relates to a
vector which comprises a frataxin (FXN) encoding nucleic acid for
use in the treatment a cardiomyopathy associated with Friedreich
ataxia in a symptomatic subject in need thereof.
Non Viral Vectors
[0059] 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
[0060] 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 proteins which mediate cell transduction.
[0061] 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 replication and
expression of transferred replicons (e.g. episomes), or integration
of transferred genetic material into the genomic DNA of host
cells.
[0062] Examples of viral vector include but are not limited to
adenoviral, retroviral, lentiviral, herpesvirus and
adeno-associated virus (AAV) vectors.
[0063] Such recombinant viruses may be produced 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 PA317 cells, PsiCRIP cells, GPenv+
cells, 293 cells, etc. Detailed protocols for producing such
replication-defective recombinant viruses may be found for instance
in WO95/14785, WO96/22378, U.S. Pat. Nos. 5,882,877, 6,013,516,
4,861,719, U.S. Pat. No. 5,278,056 and WO94/19478.
[0064] In one embodiment, adeno-associated viral (AAV) vectors are
employed.
[0065] In other embodiments, the AAV vector is AAV1, AAV2, AAV3,
AAV4, AAS, AAV6, AAV7, AAV8, AAV9, AAVrh10 or any other serotypes
of AAV that can infect humans, monkeys or other species.
[0066] In an exemplary embodiment, the AAV vector is an
AAVrh10.
[0067] By an "AAV vector" is meant a vector derived from an
adeno-associated virus serotype, including without limitation,
AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV6, etc. AAV vectors 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 functional 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 include at least those sequences
required in cis for replication and packaging (e. g., functional
ITRs) of the virus. The ITRs need not be the wild-type nucleotide
sequences, and may be altered, e. g by the insertion, deletion or
substitution of nucleotides, so long as the sequences provide for
functional rescue, replication and packaging. AAV expression
vectors are constructed using known techniques to at least provide
as operatively linked components in the direction of transcription,
control elements including a transcriptional initiation region, the
DNA of interest (i.e. the FXN gene) and a transcriptional
termination region.
[0068] The control elements are selected to be functional 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 inverted
terminal repeats" or "AAVITRs" is meant the art-recognized regions
found at each end of the AAV genome which function 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
nucleotide sequence interposed between two flanking ITRs into a
mammalian cell genome. The nucleotide sequences of AAV ITR regions
are known. See, e.g., Kotin, 1994; Berns, K I "Parvoviridae and
their Replication" 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
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 vector need not necessarily 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 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
necessarily 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.
[0069] Particularly preferred are vectors derived from AAV
serotypes having tropism for and high transduction efficiencies in
cells of the mammalian myocardium, particularly cardiomyocytes and
cardiomyocyte progenitors. A review and comparison of transduction
efficiencies of different serotypes is provided in Cearley C N et
al., 2008. In other non-limiting examples, preferred vectors
include vectors derived from any serotypes like AAV1, AAV2, AAV3,
AAV4, AAS, AAV6, AAV7, AAV8, AAV9, or AAVrh10, which have also been
shown to transduce cells of cardiomyocytes.
[0070] The selected nucleotide sequence is operably linked to
control elements that direct the transcription or expression
thereof in the subject in vivo. Such control elements can comprise
control sequences normally associated with the selected gene.
[0071] Alternatively, heterologous control sequences can be
employed. Useful heterologous control sequences generally include
those derived from sequences encoding mammalian or viral genes.
Examples include, but are not limited to, the phophoglycerate
kinase (PKG) promoter, CAG, MCK (muscle creatine kinase), the SV40
early promoter, mouse mammary tumor virus LTR promoter; adenovirus
major late promoter (Ad MLP); a herpes simplex virus (HSV)
promoter, a cytomegalovirus (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, Calif.).
[0072] Examples of heterologous promoters include the CMV
promoter.
[0073] Examples of inducible promoters include DNA responsive
elements for ecdysone, tetracycline, hypoxia andaufin.
[0074] The AAV expression vector which harbors the DNA molecule of
interest 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 therefrom.
Other portions of the AAV genome can also be deleted, so long as a
sufficient portion of the ITRs remain to allow for replication and
packaging functions. Such constructs can be designed using
techniques well known in the art. See, e. g. U.S. Pat. Nos.
5,173,414 and 5,139,941; International Publications Nos. WO
92/01070 (published 23 Jan. 1992) and WO 93/03769 (published 4 Mar.
1993); Lebkowski et al., 1988; Vincent et al., 1990; Carter, 1992;
Muzyczka, 1992; Kotin, 1994; Shelling 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 fused 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. Pat. No. 5,139,941.
In particular, several AAV vectors are described therein which are
available from the American Type Culture Collection ("ATCC") under
Accession Numbers 53222, 53223, 53224, 53225 and 53226.
Additionally, chimeric genes can be produced synthetically to
include AAV ITR sequences arranged 5' and 3' of one or more
selected nucleic acid sequences. Preferred 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; Sambrook et al. (1989) Molecular Cloning,
a laboratory manual, Cold Spring Harbor Laboratories, New York,
Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier,
and Chu et al., 1981. Particularly suitable transfection methods
include calcium phosphate co-precipitation (Graham et al., 1973),
direct microinjection into cultured cells (Capecchi, 1980),
electroporation (Shigekawa et al., 1988), liposome mediated gene
transfer (Mannino et al., 1988), lipid-mediated transduction
(Felgner et al., 1987), and nucleic acid delivery using
high-velocity microprojectiles (Klein et al., 1987).
[0075] For instance, a preferred viral vector, such as the AAVrh10,
comprises, in addition to a FXN encoding nucleic acid sequence, the
backbone of AAV vector with ITR derived from AAV-2, the promoter,
such as the mouse PGK (phosphoglycerate kinase) gene or the
cytomegalovirus/.beta.-actin hybrid promoter (CAG) consisting of
the enhancer from the cytomegalovirus immediate gene, the promoter,
splice donor and intron from the chicken .beta.-actin gene, the
splice acceptor from rabbit .beta.-globin, or any promoter such as
PGK, CAG, MCK.
Delivery of the Vectors
[0076] It is herein provided a method for treating cardiomyopathy
due to energy failure in a subject, said method comprising:
[0077] (a) providing a vector as defined above, which comprises a
nucleic acid sequence of a gene that can restore energy failure;
and
[0078] (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.
[0079] In a particular embodiment, it is herein provided a method
for treating cardiomyopathy associated with Friedreich ataxia in a
subject, said method comprising:
[0080] (a) providing a vector as defined above, which comprises a
FXN encoding nucleic acid; and
[0081] (b) delivering the vector to the subject in need thereof and
whereby FXN is expressed by the transduced cells at a
therapeutically effective level.
[0082] 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.
[0083] Additional routes of administration may also comprise local
application of the vector under direct visualization, e.g.,
superficial cortical application, or other nonstereotactic
application. The vector may be delivered intrathecally, in the
ventricles or by intravenous injection.
[0084] The target cells of the vectors of the present invention are
cells of the myocardium of a subject afflicted with a
cardiomyopathy associated with Friedreich ataxia. Preferably the
subject is a human being, adult or child.
[0085] 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, nonhuman primates and mammals of the orders Rodenta
(mice, rats, rabbit, hamsters), Carnivora (cats, dogs), and
Arteriodactyla (cows, pigs, sheep, goats, horses) as well as any
other non-human system (e. g. zebrafish model system).
[0086] 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.
Suitable 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 replenishers, electrolyte
replenishers (such as those based on Ringer's dextrose), and the
like.
[0087] Preservatives and other additives may also be present such
as, for example, antimicrobials, antioxidants, chelating agents,
and inert gases and the like.
[0088] 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.
[0089] The preferred doses and regimen may be determined by a
physician, 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 sequence encoding an FXN polypeptide using a viral expression
vector, each unit dosage of FXN polypeptide expressing vector may
comprise 2.5 to 100 .mu.l of a composition including a viral
expression vector in a pharmaceutically acceptable fluid at a
concentration ranging from 10.sup.11 to 10.sup.16 viral genome per
ml for example.
[0090] 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 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.
[0091] In a particular embodiment, the invention relates to a
vector which comprises a FXN encoding nucleic acid for use in
treatment or prevention of cardiomyopathy associated with
Friedreich ataxia in a subject wherein the vector is delivering the
subject in need thereof and wherein FXN is expressed by the
transduced cells at a therapeutically effective level.
[0092] In a particular embodiment, the invention relates to a
vector which comprises a FXN encoding nucleic acid for reversing
symptoms of cardiomyopathy associated with Friedreich ataxia in a
subject 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
[0093] A second object of the invention concerns a pharmaceutical
composition for preventing or treating cardiomyopathy due to energy
failure in a subject in need thereof, which comprises a
therapeutically effective amount of a vector which comprises a
nucleic acid sequence of a gene that can restore energy
failure.
[0094] 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.
[0095] By a "therapeutically effective amount" is meant a
sufficient amount of the vector of the invention to treat a
cardiomyopathy associated with Friedreich ataxia at a reasonable
benefit/risk ratio applicable to any medical treatment.
[0096] It will be understood that the single dosage or the total
daily dosage of the compounds and compositions of the present
invention will be decided 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 specific compound
employed; the specific composition employed, the age, body weight,
general health, sex and diet of the patient; the time of
administration, route of administration, and rate of excretion of
the specific compound employed; the duration 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 therapeutic effect and to gradually increase
the dosage until the desired 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 condition with the
number of viral or non-viral particles and/or pharmaceutical
compositions of the invention, will depend on numerous 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.
[0097] 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.
[0098] 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 administration form, as a mixture with conventional
pharmaceutical supports, to animals and human beings.
[0099] Preferably, 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 disodium 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.
[0100] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions; formulations 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
fungi.
[0101] Solutions comprising compounds of the invention 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 polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations contain a
preservative to prevent the growth of microorganisms.
[0102] The vector according to the invention can be formulated into
a composition in a neutral or salt form. Pharmaceutically
acceptable salts include the acid addition salts (formed with the
free amino groups of the 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 derived from inorganic bases such as, for example, sodium,
potassium, ammonium, calcium, or ferric hydroxides, and such
organic bases as isopropylamine, trimethylamine, histidine,
procaine and the like.
[0103] The carrier can also be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene 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 antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
many 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 delaying absorption, for example, aluminium
monostearate and gelatin.
[0104] Sterile injectable 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
required, followed 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 injectable 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 from a previously sterile-filtered solution thereof.
[0105] 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 release capsules and the like
can also be employed.
[0106] Multiple doses can also be administered.
[0107] In another embodiment, the invention relates to a
pharmaceutical composition for treating or preventing diseases
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.
[0108] The invention will be further illustrated by the following
figures and examples. However, these examples and figures should
not be interpreted in any way as limiting the scope of the present
invention.
FIGURES
[0109] FIGS. 1A-1C. Administration of AAVrh10.CAG-hXN vector at 3
weeks of age prevents the onset of cardiac failure and rescues
survival in pre-symptomatic MCK mice at a dose of 5.times.10.sup.13
vg/kg. (FIG. 1A) Survival rates of wild-type (black solid line),
treated (grey dotted line) and untreated (black dotted line) MCK
mice. n=9-10 for each group. (FIGS. 1B-1C) Relative quantification
of atrial natriuretic peptide (ANP), brain natriuretic peptide
(BNP) and sarcoplasmic reticulum Ca2+ ATPase (Serca2a) mRNA
expressions in heart at 8 and 35 weeks for wild-type (white),
treated (grey) and untreated (black) 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.
[0110] FIGS. 2A-2F. Administration of AAVrh10.CAG-hXN vector, at a
dose of 5.times.10.sup.13 vg/kg, at 7 weeks of age in symptomatic
MCK mice with severe cardiac failure reverses the cardiac
contractile dysfunction, Fe--S cluster proteins, and cardiomyocyte
and mitochondrial ultrastructure disorganization. (FIGS. 2A-2B)
Longitudinal echocardiographic assessment of the left ventricle
mass (LVM, left) and the shortening fraction (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=9-11 for each group. (FIG. 2C) 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.
(FIGS. 2D-2E) Relative quantification of atrial natriuretic peptide
(ANP), and brain natriuretic peptide (BNP) and sarcoplasmic
reticulum Ca.sup.2+ ATPase (Serca2a) mRNA expressions in heart at
8, 15 and 22 weeks for wild-type (white), treated (grey) and
untreated (black) MCK mice; n=3-5 per group and per age
(*P<0.05; **P<0.01; ns: statistically non-significant). 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. (FIG. 2F) Biochemical measurements of combined
cytosolic and mitochondrial aconitases (Aco) and succinate
dehydrogenase (SDH, complex II) 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<0.01).
Isocitrate dehydrogenase (IDH) activity was used to normalize SDH
and aconitase activites for total mitochondrial content.
[0111] FIGS. 3A-3G. Treatment of MCK mice at 5 weeks with 10 fold
decreased dose of vector delays the onset of cardiac failure.
(FIGS. 3A-3D) Echocardiographic assessment of the left ventricle
shortening fraction (FIG. 3A), ejection fraction (FIG. 3B), cardiac
output (FIG. 3C) and left ventricle mass reported to body weight
(FIG. 3D) for MCK mice treated with a dose of 5.times.10.sup.13
vg/kg (.quadrature., n=3) or 5.times.10.sup.12 vg/kg (.diamond.,
n=3), as well as for control wild-type (*, n=4) and untreated MCK
(.largecircle., n=10) mice. (FIGS. 3E-3G) Kinetic of individual
echocardiographic assessment of left ventricular shortening
fraction for MCK mice treated with 5.times.10.sup.13 vg/kg (FIG.
3E) or 5.times.10.sup.12 vg/kg (FIGS. 3F-3G).
TABLE-US-00002 TABLE 1 Echocardiographic parameters, percentage of
SDH positive cardiomyocytes and vector genome copy per individual
mice. Echocardiographic Heart Vector parameters surface copies Dose
of Shortening Cardiac LV SDH per vector Fraction Output Mass
positive diploid (vg/Kg) Mice (%) (mL/min) (10.sup.-5) (%) genome 5
.times. 10.sup.13 1 35 100 192 89 11.1 2 32 100 219 77 21.3 3 20 97
265 82 11.5 2.5 .times. 10.sup.13 1 31 67 198 85 2.9 2 25 91 278 79
7.9 1 .times. 10.sup.13 1 28 52 239 53 0.6 2 30 80 213 55 N.D. 3 33
116 210 88 3.6 5 .times. 10.sup.12 1 17 28 410 44 0.1 2 21 39 454
48 0.2 3 33 85 223 58 0.7 WT mice 1 36 102 191 87 0.0 2 36 124 227
86 0.0 3 34 137 195 94 0.0 Abbreviations: vg/kg, vector genome per
kilogram; LV, left ventricle; SDH, Succinate dehydrogenase; N.D.,
not determined. Values are presented as mean of experimental
replicate of individual measure.
EXAMPLES
Example 1
[0112] Material & Methods
[0113] Adeno-associated viral vector construction and production
Human frataxin (hFXN) cDNA, including the mitochondrial targeting
sequence, fused to a HA tag was subcloned in a pAAV2-CAG plasmid
(Sondhi, Hackett et al. 2007) to produce pAAV2-CAG-hFXN that
included the viral inverted terminal repeat (ITR) from AAV2; the
cytomegalovirus/.beta.-actin hybrid promoter, consisting of the
enhancer from the cytomegalo-virus immediate-early gene, the
promoter, splice donor, and intron from the chicken .beta.-actin
gene, and the splice acceptor from rabbit .beta.-globin. The
AAVrh10.CAG-hFXN-HA vector was produced as described earlier
(Rabinowitz, Rolling et al. 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.times.10.sup.12 vg/ml and
2.15.times.10.sup.13 vg/ml, respectively.
[0114] Animal Procedures
[0115] Mice with a specific deletion of Fxn gene in cardiac and
skeletal muscle (MCK-Cre-FxnL3/L-) (MCK mice) in 100% C57BL/6J
background were generated and genotyped as previously described
(Puccio, Simon et al. 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
wild-type mice were anesthetized by intraperitoneal injection of
ketamine/xylazine (75/10 mg/kg) to allow intravenous administration
by retro-orbital injection of AAVrh10.CAG-FXN at a dose of
5.times.10.sup.13 vg/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, respectively) to
allow intravenous administration by retro-orbital injection of
AAVrh10.CAG-FXN at a dose of 5.times.10.sup.13 vg/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 function 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 CO2 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 perfused with cooled saline
solution. For histological analysis of dorsal root ganglia, spinal
cord and cardiac tissue was embedded in OCT Tissue Tek (Sakura
Finetechnical, Torrance, Calif.) 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
ventricle and its apex were collected, then fixed and embedded in
Epon as previously described (Puccio, Simon et al. 2001).
[0116] Histopathology, Enzyme Histochemistry and Electron
Microscopy
[0117] For histochemical analysis, 10 .mu.m cryosections were
stained either with hematoxylin and eosin (H&E), Sirius red and
Fast green to label extracellular collagen, or DAB enhanced Perls
to label iron (Fe.sup.3+) deposits (Puccio, Simon et al. 2001).
[0118] Sirius red and fast green staining: Tissue sections were
fixed with 10% paraformaldehyde in 0.1 M phosphate buffer (PBS), pH
7.4 for 10 min and then incubated with a saturated solution of
picric acid containing 0.1% Direct red 80 (Sigma) for 2 min, washed
with 0.5% glacial acetic acid solution followed by deionized water,
and subsequently incubated in 0.05% Fast Green solution for 5 min,
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 mounting medium
(Histolab Products AB).
[0119] DAB-enhanced Perls iron staining: Tissue sections were fixed
with 10% paraformaldehyde in 0.1 M phosphate buffer (PBS), pH 7.4
for 20 min and incubated in Perls solution (1% HCl, 1% Potassium
Ferrocyanide) for 30 min. Staining was enhanced by incubation in
0.025% 3'-3'-diaminobenzidine tetrahydrochloride (Sigma-Aldrich),
0.005% H.sub.2O.sub.2 in PBS buffer for 30 min, and then developed
in the same buffer. Finally, sections were dehydrated in graded
alcohols, cleared in Histosol Plus (Shandom) for 5 min and mounted
using Pertex mounting medium (Histolab Products AB).
[0120] Enzyme histochemical analyses: Succinate dehydrogenase (SDH)
and Cytochrome C Oxydase (COX) activities were performed on 10
.mu.m cryostat sections of tissues, as previously described
(Puccio, Simon et al. 2001).
[0121] 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).
[0122] Immunofluorescence and Image Acquisition
[0123] Cardiac and spinal cord tissue cryosections were fixed in 4%
PFA for 10 min, washed and then permeabilized in methanol at
-20.degree. C. for 20 min. Sections were blocked and permeabilized
at the same time with PBS, 1% NGS, 5% BSA, 0.3% Triton X-100 for 1
h at room temperature (RT) and then washed in PBS, 0.2% Tween 1%
BSA 1% NGS (PBS-TBN). Subsequently, tissues were incubated
overnight (0/N) at 4.degree. C. with the rabbit polyclonal antibody
against frataxin (FXN935)(1/250) diluted in PBS-TBN (Puccio, Simon
et al. 2001). The Alexa fluor-594 goat anti-rabbit antibody (1/500)
(Molecular Probes) was incubated for 2 h 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 0/N at 4.degree. C. in M.O.M..TM. Mouse Ig Blocking
Reagent (Vector Laboratories). Section were then incubated 0/N at
4.degree. C. with the mouse monoclonal antibody to HA tag (1/150)
(Covence) diluted in M.O.M..TM. diluent (Vector Laboratories).
After washing, sections were incubated for 1 h at RT with the goat
anti-mouse antibody conjugated to Alexa Fluor-594 nm (1/500)
(Molecular Probes) diluted in M.O.M..TM. diluent. Subsequently,
sections were washed and blocked in PBS, 0.3% Triton, 2% NGS for 1
h30 at RT, washed and incubated for 2 h at RT with the rabbit
polyclonal antibody to prohibitin (1/150) (Abcam) diluted in
PBS-BTN. The Alexa Fluor-488 nm goat anti-rabbit antibody (1/500)
(Molecular Probes) was incubated 1 h30 at RT with the goat
anti-rabbit antibody conjugated to Alexa Fluor-488 nm (Molecular
Probes) diluted at 1/500 in PBS-BTN. Sections were stained with
Hoechst and mounted using Aqua-Polymount mounting medium
(Polysciences, Inc).
[0124] Confocal analysis was performed on a Leica TCS SP2 upright
confocal microsystem with a Plan Apo CS (numerical aperture 1.4)
63.times. objective. Observation of whole cardiac cryosections was
performed on a Leica Z16 APO A microsystem fitted with a
QuanteM-S125C camera and combined with a 2.times. objective (39 mm
working distance).
[0125] Quantitative Real-Time PCR Total
[0126] Total RNA was extracted from frozen heart pulverized with
the Precellys24 homogeniser (Bertin Technologies) and using TRI
Reagent (MRC) according to the manufacturer's protocol and was
treated with DNAse I treatment (Roche Biosciences). cDNA was
generated by reverse transcription using the Transcriptor first
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) with primers described in
Supplementary Table S3. 18S ribosomal RNA was used as internal
standard.
[0127] Enzyme Activities
[0128] 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).
[0129] Immunoblot Analysis
[0130] Extracts of tissues were frozen in liquid nitrogen, and then
homogenized in lysis buffer containing Tris-HCl (280 mM, pH 6.8),
10% SDS, 50% glycerol. Total protein extract (10 .mu.g or 50 .mu.g)
was analyzed on SDS-glycine polyacrylamide gels. Proteins were
transferred to nitrocellulose 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), anti-mitochondrial aconitase (R2377
purified 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 1/1,000).
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.
[0131] Statistical Analysis
[0132] All data are presented 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 Scheffe's post-hoc
test was used. A value of P<0.05 was considered significant. For
statistical comparison of two experimental groups, the bilateral
Student's t-test was used. P<0.05 was considered
significant.
[0133] Quantitative PCR on Genomic DNA
[0134] Genomic DNA was extracted from heart by using a
phenol-chloroform method. AAVrh10.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.
[0135] Results
[0136] Three week-old MCK mice that do not exhibit yet any
clinical, echocardiographic nor biochemical signs of cardiac
disease, received a single intravenous injection of
AAVrh10-CAG-hFXN at the dose of 5.4.times.10.sup.13 vg/kg (n=9).
Serial echocardiographic measurements identified that the treatment
efficiently prevented the development of the cardiac disease
associated with frataxin 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 indistinguishable from wild-type (WT) littermate animals (data
not shown). In parallel, systolic function evaluated by the
left-ventricular shortening fraction (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
molecular state of the cardiomyocytes, treated 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 peptide (ANP) and the
brain natriuretic peptide (BNP), two markers of pathology-induced
stress program induced by hemodynamic overload was markedly
increased in the heart from untreated mice at 8 weeks compared to
WT (19 and 7 times, respectively, p<0.001) (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 responsible for
diastolic Ca2+ reuptake from cytosol was reduced in untreated mice
(3.3 fold, p<0.01), treated MCK mice had normal Serca2a levels
(FIG. 1C). Histological analysis confirmed a preserved overall
heart organization in 35 week-old treated MCK mice, compared to the
myocardial degeneration with cytoplasmic vacuolization in the
necrotic cardiomyocytes observed in untreated mice at 8 weeks of
age (data not shown). Furthermore, Sirius-red staining (data not
shown) and collagen type I and III mRNA expression (data not shown)
indicated the absence of myocardial post-necrotic fibrosis in
treated animals, in comparison to the massive interstitial fibrosis
present in untreated MCK mice at 8 weeks (data not shown).
[0137] Intravenous injection of AAVrh10-FXN led to robust viral
transduction of the heart (20.85.+-.6.3 vg/cell) and liver, but
also of skeletal muscle and dorsal root ganglia (data not shown).
Western blot analysis using an anti-FXN antibody, which equally
detects human and mouse frataxins, demonstrated a significant
overexpression (>10 fold) of AAVrh10-encoded 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 shown). Co-localization with prohibitin
demonstrated the expected mitochondrial localization of human
frataxin (data not shown).
[0138] In line with the essential function of frataxin in
regulating cellular 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 mitochondrial aconitase (mAco) and succinate dehydrogenase
(SDH) (41.3% and 79.8%, respectively) (data not shown), treated
mice presented levels of activities similar to WT littermates.
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 (data not shown). 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 .alpha.-ketoglutarate dehydrogenase (KGDH) and
pyruvate dehydrogenase (PDH) in treated animals in comparison to
untreated animals (data not shown). Consistent with the absence of
Fe--S cluster deficit, no cellular iron accumulation was observed
in the cardiac tissue of treated mice (data not shown).
Furthermore, we did not detect any sign of cellular iron
homeostasis perturbation in treated animals (data not shown).
Finally, electron microscopy analysis demonstrated a normal
sarcomere organization of the cardiomyocytes and mitochondria
ultrastructure in treated mice. Untreated animals showed sparse
atrophied myofibrils and massive mitochondrial proliferation with
abnormal collapsed or swollen cristae and iron accumulation (data
not shown). All together, these data indicate that human frataxin
gene transfer using AAVrh10 in pre-symptomatic MCK mice prevented
the development of the mitochondrial FRDA cardiomyopathy at the
molecular, cellular and physiological level.
[0139] While preventing the onset of the cardiomyopathy is an
important step, at a clinical point of view it appears crucial to
determine the therapeutic potential of this gene therapy approach
when cardiac dysfunction is already present. Mutant MCK mice were
intravenously injected with AAVrh10-CAG-hFXN at the dose of
5.4.times.10.sup.13 vg/kg (n=9) at 7 weeks, when the ventricular
remodeling and left ventricular systolic 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% ejection fraction and a
decrease in LV hypertrophy and dilation in the treated mutant mice,
whereas untreated animals presented typical signs of heart failure
(FIGS. 2A-2B). Echocardiographic parameters regarding cardiac
function progressively improved to reach WT values at 11-12 weeks
of age, demonstrating a complete recovery of the ventricular
systolic function and anatomy. The survival of the mice was
significantly prolonged until at least 18 weeks of age (FIG. 2C).
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 pathology-induced stress program induced by
hemodynamic overload, reflected by the expression of ANP and BNP,
was significantly decreased one week after injection (8 weeks) in
treated mice (FIG. 2D). 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 expression of Serca2a progressively increased in
treated mice between 8 and 22 weeks, indicating that diastolic Ca2+
transport was likely restored (FIG. 2E). 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. 2F). At 22 weeks, some rare patches
with low SDH activity were detected in the cardiac tissue of
treated mice (data not shown), corresponding to fibrotic scar
probably already present at the time of treated. Interestingly,
collagen staining and expression (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
incomplete recovery of the biochemical phenotype one week after
treatment, the mitochondria in the treated animals showed some
signs of pathology, with the presence of some swollen mitochondria
presenting parallel stacks of cristae membranes (data not shown).
However, by 22 weeks, sarcomeres and mitochondria organizations
completely recovered with no sign of pathological change. All
together, these data indicate that AAVrh10.CAG-hFXN treatment in
symptomatic MCK mice resulted in a rapid clinical,
echocardiographic and biochemical improvement with a complete
correction of the FRDA cardiomyopathy.
CONCLUSION
[0140] Our data demonstrates that AAVrh10-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 reverse heart failure and cardiac
remodelling. The correction is extremely rapid and efficient, with
a striking reversal of the mitochondrial abnormalities and
biochemical Fe--S proteins deficit one week after treatment.
Despite the severity of cardiac insufficiency at the time of
treatment, the cardiac recovery is rapidly progressive, reaching
normality within 4-5 weeks of treatment.
[0141] Indeed, the correction of mitochondrial dysfunction 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 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
dysfunction is relevant to a wide variety of diseases, including
heart diseases.
[0142] In conclusion, delivery of a vector encoding hFXN in a
mammalian model of FRDA cardiomyopathy resulted in i) prevention of
the development of disease symptoms in asymptomatic individuals and
ii) reversal of disease symptoms in individuals who already
exhibited cardiomyopathy, biochemical Fe--S cluster impairment,
mitochondrial dysfunction and interstitial cardiac fibrosis.
[0143] 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 AAVrh10.CAG-FXN
Vector Injected Inducing a Detectable Level of Cardiac Transgene
Expression
[0144] Material & Methods
[0145] The dose-response evaluation was conducted 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 committee for Animal
Care and Use (Com'Eth 2012-016). Briefly, mice were anesthetized
and then injected retro-orbitaly with 1004 of AAVrh10.CAG-FXN
vector solution diluted in saline solution, for a final
injected-dose of 5.times.10.sup.13, 2.5.times.10.sup.13,
1.times.10.sup.13 and 5.times.10.sup.12 vg/kg, respectively (Table
1). Age matched WT mice were injected with 1004 of saline solution,
as control. Survival, growth and cardiac function were evaluated
weekly up to 12 weeks of age (FIGS. 3A-3D). 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 1-2 mm 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 samples were cut
alternatively at 5 and 100 .mu.m from the apex to the base of the
heart. H&E staining as well as SDH and COX activities were
performed on 5 .mu.m heart sections, at the level of the apex,
middle and base of the left ventricle. Imaging of whole heart
sections was performed at high magnification with the Hamamatsu
NanoZoomer 2.0 slide scanner and analysis were performed using
ImageJ following the color threshold method. RNA and DNA were
extracted from 100 .mu.m 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.
[0146] Results
[0147] The dose of 5.times.10.sup.13 vg/kg used in the original
study allows transduction of near 100% of the cardiomyocytes, a
transduction efficiency that will very unlikely be achieved in
humans. Therefore, to determine the lower therapeutic threshold
that is required 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
5.times.10.sup.13, 2.5.times.10.sup.13, 1.times.10.sup.13, and
5.times.10.sup.12 vg/kg (n=2-3 per dose), respectively, at 5-weeks
of age. Serial echocardiographic measurements were performed weekly
up to 12 weeks of age. Although some variability was observed, MCK
mice injected at the highest doses (5.times.10.sup.13,
2.5.times.10.sup.13 and 1.times.10.sup.13 vg/kg) did not show any
hemodynamic nor morphological anomaly up to 12 weeks of age (FIGS.
3A-3D, Table 1), demonstrating efficient correction of the early
stage systolic dysfunction and prevention of cardiac failure onset
associated with FXN deficiency. At the lower dose,
5.times.10.sup.12 vg/kg, while the hemodynamic and morphological
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 (FIGS. 3A-3B)
accompanied by a left ventricular remodeling (FIG. 3D) was observed
starting 10 weeks of age leading to a rapid declined of the cardiac
output (FIG. 3C).
[0148] 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 between
the vector genome copy, the percentage of SDH positive 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 vg/cell, with 50-60% of SDH positive
cardiomyocytes.
Example 3: Efficient Transduction of Monkey and Pig Cardiomyocytes
after Intramyocardial Injection of AAVrh10 Vector
[0149] Material & Methods
[0150] AAVrh10-CAG-GFP vector was administered using an identical
cardiac surgery procedure in monkey and pig. Following general
anesthesia, a median sternotomy was performed. The AAVrh10-CAG-GFP
vector was administered directly to the myocardium of the left
ventricle as subepicardial injections. In monkey, 9 subepicardial
injections were performed, with 3 subepicardial injections 0.5 cm
apart in 3 spots (1.66.times.10.sup.10 vg of vector per injection,
40 .mu.l/injection). The total dose of injected vector per spot was
5.times.10.sup.10 vg. In pig, 18 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.times.10.sup.10 vg (as in
monkey) of vector diluted in 40 .mu.l of PBS was injected at each
site. In the 5 other spots, vector injection was made 1 cm apart
with 8.3.times.10.sup.10 vg/injection in 3 spots and
1.25.times.10.sup.11 vg/injection in 2 spots. The total dose of
injected vector per spot was therefore 5.times.10.sup.10 vg (1
spot), 2.5.times.10.sup.11 vg (3 spots) and 3.75.times.10.sup.11 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 after vector injection was normal,
showing no changes in respect to cardiac parameters observed
immediately prior to cardiac surgery.
[0151] Monkey and pig were euthanized 3 weeks after vector
injection. The left ventricle was dissected from the remaining part
of the heart and trimmed in 4 (monkey) or 10 (pig) separate pieces
along antero-longitudinal axis. Each piece of left 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 targeting the ITR2 of AAVrh10-CAG-GFP vector and the
endogenous albumin gene. Vector copy number was assessed using the
cycle (Ct) threshold to detect albumin or ITR2 sequences. At
autopsy, there was no macroscopic lesion of left 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 myocardium
of vector-injected pig.
[0152] Results
[0153] Intravenous injection of AAVrh10-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 T K et al, Hum Gene Ther, 2:203-208, 2013). The
transduction efficacy of AAV 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 similar to human. To answer to these important pre-clinical
steps towards a gene therapy trial in patients 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 similar to
humans.
[0154] AAVrh10-CAG-GFP vector was directly 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 5.times.10.sup.10 vector
genome (vg) per spot, GFP expression 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 mm3. The mean vector copy number/cell was
1.2.
[0155] AAVrh10-CAG-GFP vector was then directly delivered in the
myocardium of a pig through epicardial injections. Six separate
spots of myocardium were injected with 3 vector injections per
spot. The spots were injected with different doses of vector:
5.times.10.sup.10 vg (spot#1), 2.5.times.10.sup.11 vg (spots#2, 3
and 4) and 3.75.times.10.sup.11 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 spots#2,
3 and 4. Injection of higher dose of AAVrh10-CAG-GFP vector in
spots#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 mm.sup.3. The mean vector copy number/cell in
spots #2 to 4 was 1.8.
[0156] Altogether, these results demonstrate that cardiomyocytes of
monkey and pig are very well transduced by AAVrh10 vector following
the direct administration of the vector to the myocardium. Results
obtained in monkey indicates that 30 injections of higher dose of
AAVrh10 vector should be sufficient to transduce >50% of
cardiomyocytes of the left ventricle in human. Thirty epicardial
viral vector injections following mini-thoracotomy has proven to be
feasible (1-hour surgical procedure) and safe in humans (Rosengart
T K et al, Hum Gene Ther, 2:203-208, 2013). Results of transduction
efficacy with AAVrh10 in pig cardiomyocytes indicates that this
porcine animal model will be suitable to evaluate the total dose of
AAVrh10-CAG-hFXN vector that it will be needed to inject in the
left ventricle myocardium of FRDA patients to express frataxin in
>50% of their cardiomyocytes.
REFERENCES
[0157] Throughout this application, various references, including
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of the art to which this invention pertains. The disclosures of
these references are hereby incorporated by reference in entirety
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Sequence CWU 1
1
217168DNAHomo sapiens 1agtctccctt gggtcagggg tcctggttgc actccgtgct
ttgcacaaag caggctctcc 60atttttgtta aatgcacgaa tagtgctaag ctgggaagtt
cttcctgagg tctaacctct 120agctgctccc ccacagaaga gtgcctgcgg
ccagtggcca ccaggggtcg ccgcagcacc 180cagcgctgga gggcggagcg
ggcggcagac ccggagcagc atgtggactc tcgggcgccg 240cgcagtagcc
ggcctcctgg cgtcacccag cccagcccag gcccagaccc tcacccgggt
300cccgcggccg gcagagttgg ccccactctg cggccgccgt ggcctgcgca
ccgacatcga 360tgcgacctgc acgccccgcc gcgcaagttc gaaccaacgt
ggcctcaacc agatttggaa 420tgtcaaaaag cagagtgtct atttgatgaa
tttgaggaaa tctggaactt tgggccaccc 480aggctctcta gatgagacca
cctatgaaag actagcagag gaaacgctgg actctttagc 540agagtttttt
gaagaccttg cagacaagcc atacacgttt gaggactatg atgtctcctt
600tgggagtggt gtcttaactg tcaaactggg tggagatcta ggaacctatg
tgatcaacaa 660gcagacgcca aacaagcaaa tctggctatc ttctccatcc
agtggaccta agcgttatga 720ctggactggg aaaaactggg tgtactccca
cgacggcgtg tccctccatg agctgctggc 780cgcagagctc actaaagcct
taaaaaccaa actggacttg tcttccttgg cctattccgg 840aaaagatgct
tgatgcccag ccccgtttta aggacattaa aagctatcag gccaagaccc
900cagcttcatt atgcagctga ggtctgtttt ttgttgttgt tgttgtttat
tttttttatt 960cctgcttttg aggacagttg ggctatgtgt cacagctctg
tagaaagaat gtgttgcctc 1020ctaccttgcc cccaagttct gatttttaat
ttctatggaa gattttttgg attgtcggat 1080ttcctccctc acatgatacc
ccttatcttt tataatgtct tatgcctata cctgaatata 1140acaaccttta
aaaaagcaaa ataataagaa ggaaaaattc caggagggaa aatgaattgt
1200cttcactctt cattctttga aggatttact gcaagaagta catgaagagc
agctggtcaa 1260cctgctcact gttctatctc caaatgagac acattaaagg
gtagcctaca aatgttttca 1320ggcttctttc aaagtgtaag cacttctgag
ctctttagca ttgaagtgtc gaaagcaact 1380cacacgggaa gatcatttct
tatttgtgct ctgtgactgc caaggtgtgg cctgcactgg 1440gttgtccagg
gagacctagt gctgtttctc ccacatattc acatacgtgt ctgtgtgtat
1500atatattttt tcaatttaaa ggttagtatg gaatcagctg ctacaagaat
gcaaaaaatc 1560ttccaaagac aagaaaagag gaaaaaaagc cgttttcatg
agctgagtga tgtagcgtaa 1620caaacaaaat catggagctg aggaggtgcc
ttgtaaacat gaaggggcag ataaaggaag 1680gagatactca tgttgataaa
gagagccctg gtcctagaca tagttcagcc acaaagtagt 1740tgtccctttg
tggacaagtt tcccaaattc cctggacctc tgcttcccca tctgttaaat
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acctaacagg 1860ctagttctaa ttccctattg ggtagatgag gggatgacaa
agaacagttt ttaagctata 1920taggaaacat tgttattggt gttgccctat
cgtgatttca gttgaattca tgtgaaaata 1980atagccatcc ttggcctggc
gcggtggctc acacctgtaa tcccagcact tttggaggcc 2040aaggtgggtg
gatcacctga ggtcaggagt tcaagaccag cctggccaac atgatgaaac
2100cccgtctcta ctaaaaatac aaaaaattag ccgggcatga tggcaggtgc
ctgtaatccc 2160agctacttgg gaggctgaag cggaagaatc gcttgaaccc
agaggtggag gttgcagtga 2220gccgagatcg tgccattgca ctgtaacctg
ggtgactgag caaaactctg tctcaaaata 2280ataataacaa tataataata
ataatagcca tcctttattg tacccttact gggttaatcg 2340tattatacca
cattacctca ttttaatttt tactgacctg cactttatac aaagcaacaa
2400gcctccagga cattaaaatt catgcaaagt tatgctcatg ttatattatt
ttcttactta 2460aagaaggatt tattagtggc tgggcatggt ggcgtgcacc
tgtaatccca ggtactcagg 2520aggctgagac gggagaattg cttgacccca
ggcggaggag gttacagtga gtcgagatcg 2580tacctgagcg acagagcgag
actccgtctc aaaaaaaaaa aaaaggaggg tttattaatg 2640agaagtttgt
attaatatgt agcaaaggct tttccaatgg gtgaataaaa acacattcca
2700ttaagtcaag ctgggagcag tggcatatac ctatagtccc agctgcacag
gaggctgaga 2760caggaggatt gcttgaagcc aggaattgga gatcagcctg
ggcaacacag caagatccta 2820tctcttaaaa aaagaaaaaa aaacctatta
ataataaaac agtataaaca aaagctaaat 2880aggtaaaata ttttttctga
aataaaatta ttttttgagt ctgatggaaa tgtttaagtg 2940cagtaggcca
gtgccagtga gaaaataaat aacatcatac atgtttgtat gtgtttgcat
3000cttgcttcta ctgaaagttt cagtgcaccc cacttactta gaactcggtg
acatgatgta 3060ctcctttatc tgggacacag cacaaaagag gtatgcagtg
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gtccttgtgg gccagccctt caggcctatt 3180ttactttcat tttacatata
gctctaattg gtttgattat ctcgttccca aggcagtggg 3240agatccccat
ttaaggaaag aaaaggggcc tggcacagtg gctcatgcct gtaatcccag
3300cactttggga ggctgaggca agtgtatcac ctgaggtcag gagttcaaga
ccagcctggc 3360caacatggca aaatcccgtc tctactaaaa atattaaaaa
attggctggg cgtggtggtt 3420cgtgcctata atttcagcta ctcaggaggc
tgaggcagga gaatcgctgt aacctggggg 3480gtggaggttg cagtgagacg
agatcatgcc acttcactcc agcctggcca acagagccat 3540actccgtctc
aaataaataa ataaataaat aaagggactt caaacacatg aacagcagcc
3600aggggaagaa tcaaaatcat attctgtcaa gcaaactgga aaagtaccac
tgtgtgtacc 3660aatagcctcc ccaccacaga ccctgggagc atcgcctcat
ttatggtgtg gtccagtcat 3720ccatgtgaag gatgagtttc caggaaaagg
ttattaaata ttcactgtaa catactggag 3780gaggtgagga attgcataat
acaatcttag aaaacttttt tttccccttt ctattttttg 3840agacaggatc
tcactttggc actcaggctg gaggacagtg gtacaatcaa agctcatggc
3900agcctcgacc tccctgggct tgggcaatcc tcccacaggt gtgcacctcc
atagctggct 3960aatttgtgta ttttttgtag agatggggtt tcaccatgtt
gcccaggctg gtctctaaca 4020cttaggctca agtgatccac ctgcctcgtc
ctcccaagat gctgggatta caggtgtgtg 4080ccacaggtgt tcatcagaaa
gctttttcta ttatttttac cttcttgagt gggtagaacc 4140tcagccacat
agaaaataaa atgttctggc atgacttatt tagctctctg gaattacaaa
4200gaaggaatga ggtgtgtaaa agagaacctg ggtttttgaa tcacaaattt
agaatttaat 4260cgaaactctg cctcttactt gtttgtagac actgacagtg
gcctcatgtt tttttttttt 4320ttaatctata aaatggagat atctaacatg
ttgagcctgg gcccacaggc aaagcacaat 4380cctgatgtga gaagtactca
gttcatgaca actgttgttc tcacatgcat agcataattt 4440catattcaca
ttggaggact tctcccaaaa tatggatgac gttccctact caaccttgaa
4500cttaatcaaa atactcagtt tacttaactt cgtattagat tctgattccc
tggaaccatt 4560tatcgtgtgc cttaccatgc ttatatttta cttgatcttt
tgcatacctt ctaaaactat 4620tttagccaat ttaaaatttg acagtttgca
ttaaattata ggtttacaat atgctttatc 4680cagctatacc tgccccaaat
tctgacagat gcttttgcca cctctaaagg aagacccatg 4740ttcatagtga
tggagtttgt gtggactaac catgcaaggt tgccaaggaa aaatcgcttt
4800acgcttccaa ggtacacact aagatgaaag taattttagt ccgtgtccag
ttggattctt 4860ggcacatagt tatcttctgc tagaacaaac taaaacagct
acatgccagc aagggagaaa 4920ggggaaggag gggcaaagtt ttgaaatttc
atgtaaattt atgctgttca aaacgacgag 4980ttcatgactt tgtgtataga
gtaagaaatg ccttttcttt tttgagacag agtcttgctc 5040tgtcacccag
gctggagtgc agtggcacga tctgggctca ctacaacctc cgcctcctgg
5100gttcaagcaa ttctctgcct cagcctcccg agtagctggg attacaggtg
cctgccacca 5160cacccggcta atttttgtat ttttagtaga gacggggttt
caccatcatg gccaggctgg 5220tcttgaactc ctgacctagt aatccacctg
cctccgcctc ccaaagtgct gggattacag 5280gcgtgagcca ctgcacccag
ccagaaatgc cttctaatct ttggtttatc ttaattagcc 5340aggacacttg
gagtgcatcc cgaagtacct gatcagtggc ccctttggaa tgtgtaaaac
5400tcagctcact tatatccctg catccgctac agagacagaa tccaagctca
tatgttccat 5460cttctctggc tgtatagttt aaggaatgga aggcaccaga
acagatttat tgaaatgttt 5520attagctgaa gatttattta gacagttgag
gaaaacatca gcacccagca gtaaaattgg 5580ctctcaaaga ttttcttctc
ctgtggaaag tcagacctct gaggccccat ccaggtagaa 5640gtactagtgc
aagaagggcc tctgctgtcc acttgtgttt ctgtgatctg tgggaacatt
5700gttaacgcca catcttgacc tcaaattgtt tagctcctgg ccagacacgg
tggctcacac 5760ctgtaatccc agcactttga gaggctgagg caggtggatc
acctgaggtt aggagttcga 5820ggccagcctg gtcaacatgg taaaaccccg
cctctactaa aaatacaaaa attagctggc 5880cgtagtggcg cacgcctgtt
atcccagcta ctcgggaggc tgaggcagga gaattgcttg 5940aacctgggtg
gtggaggttg cagtgagccg agattacacc actgcactcc agcctgggtg
6000acaagaggga aactccatta aaaaaatgta attcccgtgt ctgccatctt
aagtgtaaag 6060gtggctaaat tatatagaaa aataagacaa tatcatttcc
caattacatt cctttcctac 6120cgcactctat gatgctagct gagatttttc
caaaagaaaa tggcttaaat aaaaccctaa 6180gagaaagaaa aactttaaat
ccctccaaag ctcaaaagta atagaaacag atgagtttgg 6240agtcaggatt
tctctgtaag attgcctagg ctgtgtactg cacatctcca ggtgccactg
6300ttgacagaga ttataactac aatgtgaagt gaatggtgcc actgacagtt
atgcaaaccg 6360tccagagcat agccacctga tcctgctggg attcctcttg
ccagtccatc agcagttccc 6420cttgaaagtt tcaccaaaca tcccttaaat
ctgccctctc ctgcccgtcc ccagtggagg 6480tcctcatcat ttttcacctg
catttttgca ggagctttct tatatccacc ttcctccttt 6540tctctcagcc
catcatctag ctacacagtc tccagggtaa gctttcagaa aggcaatctc
6600ttgtctgtaa aacctaagca ggaccaaggc caagtttctt agcctgaaaa
atgtgctttt 6660ctgactgaac tgttcaggca ctgactctac atataattat
gcttttctac cccctcacac 6720tcaacacttt gactccagca atcccaaatc
cccagatccc taagtgtgct gtgctatttt 6780cacgtggctc tcagacttgg
ccagtgctgt ttccattttg gtctttattc cccacatctc 6840tgcctggggg
gtagattcta ccctgaaaaa tgttcttggc acagccttgc aaactcctcc
6900tccactcagc ctctgcctgg atgcccttga ttgttccatg tcctcagcat
accatgtttg 6960tctttcccag cactgaccta ccatgtgtca cccctgcttg
gctgtacctt ccatgaggct 7020aggactatgt gtctcctttg ttgactgctg
ttgccctagc atcttgcaca gttccttgca 7080cacaattaga gctctataaa
tgtcaaataa atgtgttata attatatgtt taagatagtt 7140gttcaaataa
actctaaata accccaac 71682210PRTHomo sapiens 2Met Trp Thr Leu Gly
Arg Arg Ala Val Ala Gly Leu Leu Ala Ser Pro1 5 10 15Ser Pro Ala Gln
Ala Gln Thr Leu Thr Arg Val Pro Arg Pro Ala Glu 20 25 30Leu Ala Pro
Leu Cys Gly Arg Arg Gly Leu Arg Thr Asp Ile Asp Ala 35 40 45Thr Cys
Thr Pro Arg Arg Ala Ser Ser Asn Gln Arg Gly Leu Asn Gln 50 55 60Ile
Trp Asn Val Lys Lys Gln Ser Val Tyr Leu Met Asn Leu Arg Lys65 70 75
80Ser Gly Thr Leu Gly His Pro Gly Ser Leu Asp Glu Thr Thr Tyr Glu
85 90 95Arg Leu Ala Glu Glu Thr Leu Asp Ser Leu Ala Glu Phe Phe Glu
Asp 100 105 110Leu Ala Asp Lys Pro Tyr Thr Phe Glu Asp Tyr Asp Val
Ser Phe Gly 115 120 125Ser Gly Val Leu Thr Val Lys Leu Gly Gly Asp
Leu Gly Thr Tyr Val 130 135 140Ile Asn Lys Gln Thr Pro Asn Lys Gln
Ile Trp Leu Ser Ser Pro Ser145 150 155 160Ser Gly Pro Lys Arg Tyr
Asp Trp Thr Gly Lys Asn Trp Val Tyr Ser 165 170 175His Asp Gly Val
Ser Leu His Glu Leu Leu Ala Ala Glu Leu Thr Lys 180 185 190Ala Leu
Lys Thr Lys Leu Asp Leu Ser Ser Leu Ala Tyr Ser Gly Lys 195 200
205Asp Ala 210
* * * * *
References