U.S. patent application number 16/395055 was filed with the patent office on 2019-08-15 for mitochondrial proteins constructs and uses thereof.
The applicant listed for this patent is Yissum Research Development Company of the Hebrew University of Jerusalem Ltd.. Invention is credited to Hagar Greif, Haya Lorberboum-Galski.
Application Number | 20190248846 16/395055 |
Document ID | / |
Family ID | 55454113 |
Filed Date | 2019-08-15 |
View All Diagrams
United States Patent
Application |
20190248846 |
Kind Code |
A1 |
Lorberboum-Galski; Haya ; et
al. |
August 15, 2019 |
MITOCHONDRIAL PROTEINS CONSTRUCTS AND USES THEREOF
Abstract
Disclosed are novel fusion protein constructs comprising a
functional mitochondrial protein, that can enter mitochondria
within intact cells. Further disclosed are methods of treating
mitochondrial disorders by the disclosed fusion proteins and
compositions therefor.
Inventors: |
Lorberboum-Galski; Haya;
(Jerusalem, IL) ; Greif; Hagar; (Ness Ziona,
IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Yissum Research Development Company of the Hebrew University of
Jerusalem Ltd. |
Jerusalem |
|
IL |
|
|
Family ID: |
55454113 |
Appl. No.: |
16/395055 |
Filed: |
April 25, 2019 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14784253 |
Oct 13, 2015 |
10287331 |
|
|
PCT/IL2014/050354 |
Apr 10, 2014 |
|
|
|
16395055 |
|
|
|
|
14034224 |
Sep 23, 2013 |
8912147 |
|
|
14784253 |
|
|
|
|
61869981 |
Aug 26, 2013 |
|
|
|
61811934 |
Apr 15, 2013 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/005 20130101;
Y10S 530/826 20130101; C12N 2740/16033 20130101; C07K 2319/10
20130101; C12Y 108/01004 20130101; C07K 14/435 20130101; C07K 14/47
20130101; Y10S 530/827 20130101; A61K 9/0019 20130101; C12Y
203/03001 20130101; C07K 2319/21 20130101; A61K 38/00 20130101;
C07K 2319/07 20130101; C12N 9/1018 20130101 |
International
Class: |
C07K 14/435 20060101
C07K014/435; C07K 14/005 20060101 C07K014/005; C07K 14/47 20060101
C07K014/47; C12N 9/10 20060101 C12N009/10; A61K 9/00 20060101
A61K009/00 |
Claims
1. A fusion protein comprising an HIV-1 transactivator of
transcription (TAT) domain fused to a human pyruvate dehydrogenase
E1 component alpha subunit (PDHE1a) and a human mitochondria
targeting sequence (MTS), wherein the MTS is situated between the
TAT domain and the PDHE1a, the PDHE1a is C-terminal to the MTS, and
the MTS is heterologous to the PDHE1a.
2. The fusion protein of claim 1, wherein the MTS is a human
citrate synthase (CS) MTS.
3. The fusion protein of claim 2, wherein the MTS comprises the
amino acid sequence of SEQ ID NO. 23.
4. The fusion protein of claim 1, further comprising a linker
covalently linking the TAT domain to the MTS.
5. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and the fusion protein according to claim 1 as
an active ingredient.
6. A method for introducing a human pyruvate dehydrogenase E1
component alpha subunit (PDHE1a) into a subject, comprising
administering to the subject the fusion protein of claim 1.
7. A method for treating or alleviating a mitochondrial disorder,
comprising administering to a human subject in need thereof the
fusion protein of claim 1.
8. The method of claim 7, wherein the fusion protein is
intravenously administered to the subject.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation application of
U.S. Ser. No. 14/784,253, filed Oct. 13, 2015, which is US national
stage application of International Application PCT/IL2014/050354,
filed Apr. 10, 2014, which is a continuation-in-part of U.S. Ser.
No. 14/034,224, filed Sep. 23, 2013, now U.S. Pat. No. 8,912,147,
issued Dec. 16, 2014, which claims the benefit under 35 U.S.C.
.sctn. 119(e) of U.S. Provisional Application Ser. No. 61/869,981,
filed Aug. 26, 2013, and, which claims the benefit under 35 U.S.C.
.sctn. 119(e) of U.S. Provisional Application Ser. No. 61/811,934,
filed Apr. 15, 2013 the contents of each of which are incorporated
by reference in their entireties into the present disclosure.
SEQUENCE LISTING
[0002] The present application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Oct. 13, 2015, is named BIOB-001NO1US_ST25.txt and is 76,765
bytes in size.
TECHNOLOGICAL FIELD
[0003] Disclosed are novel fusion protein constructs comprising a
functional mitochondrial protein. Further disclosed are methods of
treating mitochondrial disorders by the disclosed fusion proteins
and compositions therefor.
PRIOR ART
[0004] References considered to be relevant as background to the
presently disclosed subject matter are listed below: [0005] [1]
Chinnery P F, Schon E A (2003) Mitochondria. J Neurol Neurosurg
Psychiatry 74: 1188-1199. [0006] [2] DiMauro S, Schon E A (2003)
Mitochondrial respiratory-chain diseases. N Engl J Med 348:
2656-2668. [0007] [3] Brautigam C A, Chuang J L, Tomchick D R,
Machius M, Chuang D T (2005) Crystal structure of human
dihydrolipoamide dehydrogenase: NAD+/NADH binding and the
structural basis of disease-causing mutations. J Mol Biol 350:
543-552. [0008] [4] Brady R O, Schiffmann R (2004)
Enzyme-replacement therapy for metabolic storage disorders. Lancet
Neurol 3: 752-756. [0009] [5] Wang D, Bonten E J, Yogalingam G,
Mann L, d'Azzo A (2005) Short-term, high dose enzyme replacement
therapy in sialidosis mice. Mol Genet Metab 85: 181-189. [0010] [6]
Luft F C (2003) Transducing proteins to manipulate intracellular
targets. J Mol Med (Berl) 81: 521-523. [0011] [7] Kabouridis P S
(2003) Biological applications of protein transduction technology.
Trends
[0012] Biotechnol 21: 498-503. [0013] [8] Green M, Loewenstein P M
(1988) Autonomous functional domains of chemically synthesized
human immunodeficiency virus tat trans-activator protein. Cell 55:
1179-1188. [0014] [9] Frankel A D, Pabo C O (1988) Cellular uptake
of the tat protein from human immunodeficiency virus. Cell 55:
1189-1193. [0015] [10] Futaki S, Suzuki T, Ohashi W, Yagami T,
Tanaka S, et al. (2001) Arginine-rich peptides. An abundant source
of membrane-permeable peptides having potential as carriers for
intracellular protein delivery. J Biol Chem 276: 5836-5840. [0016]
[11] Schwarze S R, Ho A, Vocero-Akbani A, Dowdy S F (1999) In vivo
protein transduction: delivery of a biologically active protein
into the mouse. Science 285: 1569-1572. [0017] [12] Guo X, Hutcheon
A E, Zieske J D (2004) Transduction of functionally active TAT
fusion proteins into cornea. Exp Eye Res 78: 997-1005. [0018] [13]
Del Gaizo V, MacKenzie J A, Payne R M (2003) Targeting proteins to
mitochondria using TAT. Mol Genet Metab 80: 170-180. [0019] [14]
Harding A E (1981) Friedreich's ataxia: a clinical and genetic
study of 90 families with an analysis of early diagnostic criteria
and intrafamilial clustering of clinical features. Brain 104:
589-620. [0020] [15] Schulz J B, Boesch S, Burk K, Dun A, Giunti P,
et al. (2009) Diagnosis and treatment of Friedreich ataxia: a
European perspective. Nat Rev Neurol 5: 222-234. [0021] [16] Dun A,
Cossee M, Agid Y, Campuzano V, Mignard C, et al. (1996) Clinical
and genetic abnormalities in patients with Friedreich's ataxia. N
Engl J Med 335: 1169-1175. [0022] [17] Campuzano V, Montermini L,
Lutz Y, Cova L, Hindelang C, et al. (1997) Frataxin is reduced in
Friedreich ataxia patients and is associated with mitochondrial
membranes. Hum Mol Genet 6: 1771-1780. [0023] [18] Rotig A, de
Lonlay P, Chretien D, Foury F, Koenig M, et al. (1997) Aconitase
and mitochondrial iron-sulphur protein deficiency in Friedreich
ataxia. Nat Genet 17: 215-217. [0024] [19] Lodi R, Cooper J M,
Bradley J L, Manners D, Styles P, et al. (1999) Deficit of in vivo
mitochondrial ATP production in patients with Friedreich ataxia.
Proc Natl Acad Sci USA 96: 11492-11495. [0025] [20] Delatycki M B,
Camakaris J, Brooks H, Evans-Whipp T, Thorburn D R, et al. (1999)
Direct evidence that mitochondrial iron accumulation occurs in
Friedreich ataxia. Ann Neurol 45: 673-675. [0026] [21] Tsou A Y,
Friedman L S, Wilson R B, Lynch D R (2009) Pharmacotherapy for
Friedreich ataxia. CNS Drugs 23: 213-223. [0027] [22] Perlman S L
(2012) A review of Friedreich ataxia clinical trial results. J
Child Neurol 27: 1217-1222. [0028] [23] Rapoport M, Saada A,
Elpeleg O, Lorberboum-Galski H (2008) TAT-mediated delivery of LAD
restores pyruvate dehydrogenase complex activity in the
mitochondria of patients with LAD deficiency. Mol Ther 16: 691-697.
[0029] [24] Rapoport M, Salman L, Sabag O, Patel M S,
Lorberboum-Galski H (2011) Successful TAT-mediated enzyme
replacement therapy in a mouse model of mitochondrial E3
deficiency. J Mol Med (Berl) 89: 161-170. [0030] [25] Vyas P M,
Tomamichel W J, Pride P M, Babbey C M, Wang Q, et al. (2012) A
TAT-frataxin fusion protein increases lifespan and cardiac function
in a conditional Friedreich's ataxia mouse model. Hum Mol Genet 21:
1230-1247. [0031] [26] Gakh O, Cavadini P, Isaya G (2002)
Mitochondrial processing peptidases. Biochim Biophys Acta 1592:
63-77. [0032] [27] Cavadini P, Adamec J, Taroni F, Gakh O, Isaya G
(2000) Two-step processing of human frataxin by mitochondrial
processing peptidase. Precursor and intermediate forms are cleaved
at different rates. J Biol Chem 275: 41469-41475. [0033] [28]
Schmucker S, Argentini M, Carelle-Calmels N, Martelli A, Puccio H
(2008) The in vivo mitochondrial two-step maturation of human
frataxin. Hum Mol Genet 17: 3521-3531. [0034] [29] Gakh O,
Bedekovics T, Duncan S F, Smith D Yt, Berkholz D S, et al. (2010)
Normal and Friedreich ataxia cells express different isoforms of
frataxin with complementary roles in iron-sulfur cluster assembly.
J Biol Chem 285: 38486-38501. [0035] [30] Gavel Y, von Heijne G
(1990) Cleavage-site motifs in mitochondrial targeting peptides.
Protein Eng 4: 33-37. [0036] [31] Braun H P, Schmitz U K (1997) The
mitochondrial processing peptidase. Int J Biochem Cell Biol 29:
1043-1045. [0037] [32] Horwich A (1990) Protein import into
mitochondria and peroxisomes. Curr Opin Cell Biol 2: 625-633.
[0038] [33] Saada A, Edvardson S, Rapoport M, Shaag A, Amry K, et
al. (2008) C6ORF66 is an assembly factor of mitochondrial complex
I. Am J Hum Genet 82: 32-38. [0039] [34] Cheng T L, Liao C C, Tsai
W H, Lin C C, Yeh C W, et al. (2009) Identification and
characterization of the mitochondrial targeting sequence and
mechanism in human citrate synthase. J Cell Biochem 107: 1002-1015.
[0040] [35] Santos R, Lefevre S, Sliwa D, Seguin A, Camadro J M, et
al. (2010) Friedreich ataxia: molecular mechanisms, redox
considerations, and therapeutic opportunities. Antioxid Redox
Signal 13: 651-690. [0041] [36] Bulteau A L, O'Neill H A, Kennedy M
C, Ikeda-Saito M, Isaya G, et al. (2004) Frataxin acts as an iron
chaperone protein to modulate mitochondrial aconitase activity.
Science 305: 242-245. [0042] [37] Richardson T E, Yu A E, Wen Y,
Yang S H, Simpkins J W (2012) Estrogen prevents oxidative damage to
the mitochondria in Friedreich's ataxia skin fibroblasts. PLoS One
7: e34600. [0043] [38] WO 2009/098682. [0044] [39]
http://www.curefa.org/pipeline.html. [0045] [40] Tang N, et al.
(2012) Stable Overexpression of Arginase I and Ornithine
Transcarbamylase in HepG2 Cells Improves Its Ammonia
Detoxification. Journal of Cellular Biochemistry 113: 518-527.
[0046] [41] Cunningham, S. C. et al. (2011) Induction and
Prevention of Severe Hyperammonemia in the spf.sup.ash Mouse Model
of Ornithine Transcarbamylase Deficiency Using shRNA and
rAAV-mediated Gene Delivery. Molecular Therapy 19(5):854-9.
[0047] Acknowledgement of the above references herein is not to be
inferred as meaning that these are in any way relevant to the
patentability of the presently disclosed subject matter.
BACKGROUND
[0048] Mitochondria play a major and critical role in cellular
homeostasis--they participate in intracellular signaling and
apoptosis, and perform numerous biochemical tasks, such as in
pyruvate oxidation, in the citric acid cycle (also referred to as
the Krebs cycle), and in metabolism of amino acids, fatty acids,
nucleotides and steroids. However, the most crucial task of
mitochondria is their role in cellular energy metabolism. This
includes .beta.-oxidation of fatty acids and production of ATP by
means of the electron-transport chain and the
oxidative-phosphorylation system [1, 2].
[0049] Most of the approximately 900 gene products in the
mitochondria are encoded by nuclear DNA (nDNA) while mitochondrial
DNA (mtDNA) only contains 13 protein encoding genes. Most of the
polypeptides encoded by nDNA genes are synthesized with a
mitochondrial targeting sequence (MTS), allowing their import from
the cytoplasm into mitochondria through the translocation machinery
(TOM/TIM). Upon entering the mitochondria, the MTS is recognized
and cleaved off, allowing for proper processing and, if necessary,
assembly into mitochondrial enzymatic complexes [3].
[0050] Currently, there is no cure for genetic mitochondrial
metabolic disorders and treatment is mostly palliative.
[0051] Enzyme or Protein Replacement Therapy (E/PRT) is a
therapeutic approach for metabolic disorders, whereby the deficient
or absent protein/enzyme is artificially manufactured, purified and
administered intravenously to the patient in need thereof on a
regular basis.
[0052] After many years of extensive research, E/PRT has been
successfully accepted as the treatment of choice for metabolic
lysosomal storage diseases, including Gaucher disease, Fabry
disease and attenuated variants of mucopolysaccaridoses type 1 (MPS
1). However, the inability of the intravenously administered
enzymes to penetrate the blood-brain barrier severely limits the
application of this approach for the treatment of other metabolic
disorders that involve the central nervous system (CNS) [4, 5].
[0053] One approach for delivering proteins into cells is their
fusion with protein transduction domains (PTDs), a group of short
peptides that serve as delivery vectors for large molecules.
Generally, PTDs are defined as short, water-soluble and partly
hydrophobic, and/or polybasic peptides (at most 30-35 amino acids
residues) with a net positive charge at physiological pH [6, 7].
The main feature of PTDs is that they are able to penetrate the
cell membrane at low micromolar concentrations both in vitro and in
vivo without using any chiral receptors and without causing
significant membrane damage. Furthermore, and even more
importantly, these peptides are capable of internalizing
electrostatically or covalently bound biologically active cargoes
(such as drugs) with high efficiency and with low toxicity. The
mechanism(s) by which PTDs enter the cells has not been completely
understood. One of the well-characterized PTDs is the
transactivator of transcription (TAT) peptide originating from the
HIV-1 virus. TAT is an 11-amino-acid (residues 47-57) arginine- and
lysine-rich portion of the Tat protein encoded by HIV-1 virus [8,
9]. TAT-fusion proteins have been previously shown to be rapidly
and efficiently introduced into cultured cells, intact tissue and
live tissues when injected into mice [10-12]. It has also been
demonstrated that TAT fusion proteins traverse mitochondrial
membranes [13, 38].
[0054] There has been great progress in the use of PTD-fusion
proteins for the delivery of different macromolecules into cells
both in vitro and in vivo. This system can be used even for the
delivery of cargoes into the brain across the blood-brain barrier.
In addition, the ability to target specific intracellular
sub-localizations, such as the nuclei, the mitochondria and
lysosomes, further expands the possibilities of this delivery
system to the development of sub-cellular organelle-targeted
therapy. The therapeutic applications seem almost unlimited, and
the use of the TAT-based delivery system has extended from proteins
to a large variety of cargoes such as oligonucleotides, imaging
agents, low molecular mass drugs, nanoparticles, micelles and
liposomes. As will be shown, this PTD system is used for developing
fusion constructs of functional mitochondrial proteins, for
treatment of mitochondrial disorders, for example Friedreich
ataxia.
[0055] Friedreich ataxia is an autosomal recessive degenerative
disorder characterized by ataxia, areflexia, sensory loss,
weakness, scoliosis, and cardiomyopathy. Diabetes mellitus, optic
neuropathy, and hearing loss are also seen in patients suffering
from this disease [14, 15]. Most patients with Friedreich ataxia
(97%) have expansions of a GAA repeat in the first intron on both
alleles of the gene encoding the mitochondrial protein frataxin
[15, 16] whose expression is reduced in Friedreich ataxia [17]. The
size of the GAA repeat expansion inversely correlates with frataxin
expression and with the age of disease onset [16]. A deficiency of
frataxin in cells leads to decreased activities of mitochondrial
iron-sulfur cluster-containing enzymes, to an accumulation of iron
in the mitochondrial matrix, increased sensitivity to oxidative
stress, as well as to impaired adenosine triphosphate (ATP)
production [18-20].
[0056] Current targets for disease-modifying drug development
include agents targeting the mitochondria, aimed to (1) reduce
oxidative stress and free-radical generation; (2) improve ATP
production; (3) reduce iron accumulation; and (4) increase frataxin
production and the assembly of iron-sulfur clusters [21]. There are
presently 21 agents or classes of therapeutic agents enrolled in
the research pipeline of Friedreich ataxia disease [39]. Millions
of dollars from public, private, and industry-based initiatives
have been dedicated to research of Friedreich ataxia therapeutics.
Despite this vigorous international effort, there is as yet no
proven disease-modifying therapy for Friedreich ataxia [22].
[0057] Development of E/PRT using the TAT delivery system in
mitochondrial disorders was previously reported for lipoamide
dehydrogenase (LAD) mitochondrial deficiency [23, 38]. LAD is the
E3 subunit of the three .alpha.-ketoacid dehydrogenase complexes in
the mitochondrial matrix, which are crucial for the metabolism of
carbohydrates and amino acids. These complexes are the pyruvate
dehydrogenase complex (PDHC), the .alpha.-ketoglutarate
dehydrogenase complex (KGDHC) and the branched chain ketoacid
dehydrogenase complex (BCKDHC). This previously reported TAT
delivery system was based on a TAT-LAD fusion protein comprising
the natural precursor sequence of the human LAD containing the
N-terminal 35 amino acid mitochondrial targeting sequence (MTS).
The natural MTS of LAD was used to facilitate processing of the
TAT-LAD construct upon delivery into the mitochondria, thus
allowing the incorporation of the delivered LAD into the a-ketoacid
dehydrogenase complexes. This TAT-LAD construct was demonstrated to
enter patients' cells rapidly, and efficiently reaching the
mitochondria. Inside the mitochondria, TAT-LAD was shown to be
processed and to restore LAD activity [23]. Moreover, delivery of
TAT-LAD into E3-deficient mice tissues was also demonstrated [24].
In mice tissues, a single administration of TAT-LAD resulted in a
significant increase in the enzymatic activity of the mitochondrial
multienzyme complex pyruvate-dehydrogenase complex within the
liver, heart and, most importantly, brain of TAT-LAD-treated
E3-deficient mice [24].
[0058] Notably, TAT-LAD was shown to be able to restore the
activity of the pyruvate dehydrogenase complex (PDHC) within
treated patients' cells almost back to its normal levels. PDHC is a
9.5.times.10.sup.6 Da macromolecular machine whose multipart
structure assembly process involves numerous different subunits: a
pentagonal core of 60 units of the E2 component (dihydrolipoamide),
attached to 30 tetramers of the E1 component (.alpha.2.beta.2)
(pyruvate decarboxylase), 12 dimers of the E3 (LAD,
dihydrolipoamide) component and 12 units of the E3 binding protein.
The structure of all a-ketoacid dehydrogenase complexes is similar
to that of PDHC. The complexity of this structure emphasizes the
significance in showing that TAT-mediated replacement of one
mutated component restores the activity of an essential
mitochondrial multi-component enzymatic complex in cells of
enzyme-deficient patients.
[0059] Previous studies of mitochondria delivery system primarily
used the native MTS of mitochondrial proteins (e.g. LAD) and showed
that the native MTS was necessary for maximal restoration of LAD
enzymatic function. Deleting the MTS restored a significantly
smaller amount of LAD activity within the mitochondria. Since TAT
can move both ways across a membrane and thus pull the therapeutic
cargo out of the mitochondria, when MTS is included, the matrix
processing peptidases recognizes the sequence and clips it, and the
cargo (e.g. mature LAD) is left in the mitochondrial matrix while
the TAT peptide can transduce out of the mitochondrion. Repeated
dosing should therefore result in accumulating amounts of cargo in
the mitochondria over time.
[0060] A TAT-FRATAXIN (TAT-FRA, also referred to as TAT-FXN) fusion
protein for putative treatment of Friedreich's ataxia was recently
reported [25]. This TAT-FXN fusion protein was shown to bind iron
in vitro, transduce into the mitochondria of Friedreich ataxia
deficient fibroblasts and also reduce caspase-3 activation in
response to an exogenous iron-oxidant stress. In this TAT-FXN
fusion protein, the authors used the native MTS of frataxin that
consists of 80 amino acid residues (aa) for preparing their TAT-FXN
fusion protein [26].
[0061] It is known that FXN mRNA is translated to a precursor
polypeptide that is transported to the mitochondrial matrix and
processed to at least two forms, namely FXN42-210 and FXN81-210.
FXN42-210 is a transient processing intermediate, whereas FXN81-210
represents the mature protein [27, 28]. However, it was found that
both FXN42-210 and FXN81-210 are present in control cell lines and
tissues at steady-state, and that FXN42-210 is consistently more
depleted than FXN81-210 in samples from Friedreich's ataxia
patients [29].
[0062] Most nuclear-encoded mitochondrial proteins contain a
cleavable N-terminal MTS that directs mitochondrial targeting of
the protein; as detailed above, the N-terminal MTS is cleaved off
by matrix processing proteases at a well-conserved RXY .dwnarw.
(S/A) motif, which is a three amino acid (aa) motif, where X can be
any aa, followed by serine or alanine and cleavage is performed
after the three first amino acids [26, 30-31]. These N-terminal
MTSs are typically 15-30 amino acids in length including 3-5
nonconsecutive basic amino acid (arginine/lysine) residues, often
with several serine/threonine residues but without acidic amino
acid (asparate/glutamate) residues. In their molecular structure,
these MTSs are able to form strong basic amphipathic
.alpha.-helices that are essential for efficient mitochondrial
transportation [32]. Thus, by way of example, the long 80-aa native
MTS of frataxin as well as its two-step processing can reduce its
efficiency in the delivery of cargos into the matrix of the
mitochondria.
SUMMARY OF THE INVENTION
[0063] Provided is a fusion protein comprising a HIV-1
transactivator of transcription (TAT) domain, a functional human
mitochondrial protein and a human mitochondria targeting sequence
(MTS) situated between said TAT domain and said functional human
mitochondrial protein and wherein said human MTS is heterologous to
said functional human mitochondrial protein.
[0064] In the disclosed fusion protein said functional human
mitochondrial protein can be situated C-terminal to said human
MTS.
[0065] In the disclosed fusion protein said human mitochondrial
protein can be a functional human mitochondrial protein per se
and/or a component of a mitochondrial multi-component complex, for
example human frataxin and ornithine transcarbamoylase (OTC).
[0066] In the disclosed fusion protein, said MTS can comprise from
about 15 to about 40 amino acid residues, including from about 3 to
about 5 nonconsecutive basic amino acid residues, and optionally
from about 1 to about 3 or 4 or 5 serine/threonine residues.
[0067] Non-limiting examples of the MTS comprised in the disclosed
fusion protein are any one of human mitochondrial citrate synthase
MTS (the amino acid and the nucleic acid sequence encoding therefor
are denoted by SEQ ID NO. 23 and SEQ ID NO. 3, respectively), the
human lipoamide dehydrogenase MTS (the amino acid and the nucleic
acid sequence encoding therefor are denoted by SEQ ID NO. 24 and
SEQ ID NO. 5, respectively), the MTS of the human C6ORF66 gene
product (the amino acid and the nucleic acid sequence encoding
therefor are denoted by SEQ ID NO. 25 and SEQ ID NO. 4,
respectively) and the MTS of human mitochondrial GLUD2 (encoded by
the nucleic acid sequence denoted by SEQ ID NO. 16).
[0068] Further provided is a fusion protein comprising a HIV-1
transactivator of transcription (TAT) domain fused to a functional
human mitochondrial protein and a human mitochondria targeting
sequence (MTS) of a human mitochondrial protein selected from
citrate synthase (CS) and lipoamide dehydrogenase (LAD) situated
between said TAT domain and said functional human mitochondrial
protein, wherein said functional human mitochondrial protein is
C-terminal to said MTS of human lipoamide dehydrogenase or human
citrate synthase.
[0069] The disclosed fusion protein as herein defined may further
comprise a linker covalently linking said TAT domain to said MTS
sequence.
[0070] In the disclosed fusion protein, the fusion protein may have
the amino acid sequence denoted by SEQ ID NO. 30, comprising a
HIV-1 transactivator of transcription (TAT) domain having the amino
acid sequence denoted by SEQ ID NO. 27 fused to human frataxin
having the amino acid sequence denoted by SEQ ID NO. 26 and a
mitochondria targeting sequence (MTS) of human lipoamide
dehydrogenase having the amino acid sequence denoted by SEQ ID NO.
24, said MTS situated between said TAT domain and said frataxin,
and is linked to said TAT domain via a linker having the amino acid
sequence denoted by SEQ ID NO. 32, and wherein said frataxin is
C-terminal to said MTS of human lipoamide dehydrogenase.
[0071] In further embodiments of the disclosed fusion protein, the
fusion protein may have the amino acid sequence denoted by SEQ ID
NO. 28, comprising a HIV-1 transactivator of transcription (TAT)
domain having the amino acid sequence denoted by SEQ ID NO. 27
fused to human frataxin having the amino acid sequence denoted by
SEQ ID NO. 26 and a mitochondria targeting sequence (MTS) of human
citrate synthase having the amino acid sequence denoted by SEQ ID
NO. 23, said MTS situated between said TAT domain and said
frataxin, and is linked to said TAT domain via a linker having the
amino acid sequence denoted by SEQ ID NO. 32, and wherein said
frataxin is C-terminal to said MTS of human citrate synthase.
[0072] Further disclosed is a composition comprising a
physiologically acceptable carrier and as an active ingredient a
fusion protein as disclosed herein, and a pharmaceutical
composition comprising a physiologically or a pharmaceutically
acceptable carrier and as an active ingredient a fusion protein as
disclosed herein.
[0073] Further disclosed is a pharmaceutical composition for
restoring, at least in part, activity of a defective or deficient
or unfunctional human mitochondrial protein in a subject in need.
The said human mitochondrial protein can be active per se, or can
be a member of a functional mitochondrial protein complex.
[0074] Further disclosed is a composition comprising a
physiologically acceptable carrier and as an active ingredient a
fusion protein comprising a HIV-1 transactivator of transcription
(TAT) domain fused to human frataxin and a human mitochondria
targeting sequence (MTS) of a human mitochondrial protein selected
from citrate synthase (CS) and lipoamide dehydrogenase (LAD)
situated between said TAT domain and said frataxin, wherein said
frataxin is C-terminal to said MTS of human lipoamide dehydrogenase
or human citrate synthase, as disclosed herein.
[0075] The present disclosure further provides a composition
comprising as an active ingredient a fusion protein having the
amino acid sequence denoted by SEQ ID NO. 30 or a fusion protein
having the amino acid sequence denoted by SEQ ID NO. 28 and a
physiologically acceptable carrier.
[0076] The pharmaceutical composition disclosed herein can be
intended for treating or alleviating a mitochondrial disorder, such
as but not limited to Friedreich's ataxia or any other disorder
associated with deficiency of frataxin or defective frataxin or a
disorder associated with a deficiency of OTC or with defective
OTC.
[0077] Further disclosed is a pharmaceutical composition for the
treatment of Friedreich's Ataxia by intravenous administration to a
subject in need thereof, said composition comprising a
therapeutically effective amount of a fusion protein having the
amino acid sequence denoted by SEQ ID NO. 30 or a fusion protein
having the amino acid sequence denoted by SEQ ID NO. 28 and at
least one of pharmaceutically acceptable carrier, diluent, additive
and excipient.
[0078] A non-limiting example of a pharmaceutical composition as
herein defined is wherein the therapeutically effective amount
administered is from about 0.5 mg/Kg to about 2 mg/Kg body weight
of the subject.
[0079] Thus, the disclosed fusion protein can be used in a method
for the treatment of a mitochondrial disorder, such as but not
limited to Friedreich's ataxia or any other disorder associated
with deficiency of frataxin or defective frataxin or, respectively
a disorder associated with a deficiency of OTC or with defective
OTC.
[0080] Further provided is a method for treating or alleviating a
mitochondrial disorder, said method comprising the step of
administering to a subject in need of such treatment a
therapeutically effective amount of the fusion protein disclosed
herein, thereby treating a mitochondria disorder.
[0081] In some embodiments of the disclosed method, the functional
protein is frataxin, respectively OTC, and the mitochondrial
disorder is Friedreich's ataxia or any other disorder associated
with deficiency of frataxin or defective frataxin or, respectively,
a disorder associated with a deficiency of OTC or with defective
OTC.
[0082] The disclosed method for treating or alleviating a
mitochondrial disorder can further comprises administering an
additional therapeutic agent.
[0083] Further provided is a method for introducing a functional
mitochondrial protein into mitochondria of a subject, said method
comprising the step of administering to a subject in need of such
treatment a therapeutically effective amount of the fusion protein
as disclosed herein, thereby introducing a functional human
mitochondrial protein into the mitochondria of a subject in need
thereof.
[0084] In the disclosed method for introducing a functional
mitochondrial protein into mitochondria of a subject, said
introduced functional human mitochondrial protein may restore at
least partial activity of a wild type human mitochondrial protein,
at least 5%, at least 10%, at least 20%, at least 30%, at least
40%, at least 50%, at least 60%, at least 70%, at least 80%, at
least 90% or up to 100% of the activity of a wild type human
mitochondrial protein.
[0085] Further disclosed is a method for restoring, at least in
part, activity of a defective or deficient or un-functional human
mitochondrial protein in a subject in need, by administering to
said subject a therapeutically effective amount of a fusion protein
according to the present disclosure. The said human mitochondrial
protein can be active per se, or can be a member of a functional
mitochondrial protein complex.
[0086] Still further, provided is a method for alleviating
oxidative stress in a subject in need thereof, said method
comprising the step of administering to a subject in need of such
treatment a therapeutically effective amount of the fusion protein
as disclosed herein, thereby alleviating oxidative stress in said
subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0087] In order to better understand the subject matter that is
disclosed herein and to exemplify how it may be carried out in
practice, embodiments will now be described, by way of non-limiting
example only, with reference to the accompanying drawings, in
which:
[0088] FIG. 1A-1D: Schematic structures of the various TAT-MTS-FRA
fusion proteins and the expected molecular weights thereof.
Abbreviations: H, His tag; TAT, transactivator of transcription;
FRA (or fra), Frataxin; MTS, mitochondrial translocation sequence;
cs, Citrate synthase; orf, C6ORF66; lad, LAD and kDa, kilo
Dalton.
[0089] FIG. 2A-2D: Expression and sub-cellular localization of
TAT-MTSfra-FRA and of TAT-MTSorf-FRA fusion proteins
[0090] FIG. 2 presents an image of SDS-PAGE analysis of bacterial
sub-fractions expressing the TAT-MTSfra-FRA fusion protein (FIG.
2A) and an immunoblot of Western blot analysis thereof (FIG. 2B)
using anti-His antibody; an image of SDS-PAGE analysis of bacterial
sub-fractions expressing TAT-MTSorf-FRA fusion protein (FIG. 2C)
and an immunoblot of Western blot analysis thereof using anti-His
antibody is presented in FIG. 2D.
[0091] Abbreviations (for FIGS. 2A-2D): 1, whole cell extract; 2,
soluble fraction; 3, insoluble fraction; kDa, kilo Dalton; and
M=marker. Arrow heads indicate the fusion proteins.
[0092] FIG. 3A-3D: Expression and sub-cellular localization of
TAT-MTSlad-FRA and TAT-MTScs-FRA fusion proteins
[0093] FIG. 3 presents an image of SDS-PAGE analysis of bacteria
sub-fractions expressing the TAT-MTSlad-FRA fusion protein (FIG.
3A) and an immunoblot of Western blot analysis thereof (FIG. 3B)
using anti-His antibody; an image of SDS-PAGE analysis of bacterial
sub-fractions expressing TAT-MTScs-FRA fusion protein (FIG. 3C) and
an immunoblot of Western blot analysis thereof using anti-His
antibody is presented in FIG. 3D.
[0094] Abbreviations (for FIG. 3A-3D): 1, whole cell extract; 2.
soluble fraction; 3, insoluble fraction; KDa, kilo Dalton; and
M=marker. Arrow heads indicate the fusion proteins.
[0095] FIG. 4A-4D: Purification of TAT-MTSfra-FRA and of
TAT-MTSorf-FRA fusion proteins
[0096] FIG. 4 presents an image of an affinity chromatography
purification profile of the fusion protein TAT-MTSfra-FRA (FIG. 4A)
and an image of SDS-PAGE analysis of the purification steps
obtained for TAT-MTSfra-FRA is presented in FIG. 4B. An image of an
affinity chromatography purification profile of the fusion protein
TAT-MTSorf-FRA is presented in FIG. 4C and an image of SDS-PAGE
analysis of the purification steps obtained for TAT-MTSorf-FRA is
presented in FIG. 4D.
[0097] Abbreviations: M, marker; 1, whole cell extract; 2, pre-run
fraction (the soluble sub-fraction of bacterial cells expressing
the fusion protein); 3, flow through; 4, elution with 100 mM
imidazole; and 5-9, elution with 250 mM imidazole.
[0098] FIG. 5A-5D: Purification of TAT-MTSlad-FRA and of
TAT-MTScs-FRA fusion proteins FIG. 5 presents an image of an
affinity chromatography purification profile of the fusion protein
TAT-MTSlad-FRA (FIG. 5A) and an image of SDS-PAGE analysis of the
purification steps obtained for TAT-MTSlad-FRA is presented in FIG.
5B. An image of an affinity chromatography purification profile of
the fusion protein TAT-MTScs-FRA is presented in FIG. 5C and an
image of SDS-PAGE analysis of the purification steps obtained for
TAT-MTScs-FRA is presented in FIG. 5D.
[0099] Abbreviations: M, marker; 1, whole cell extract; 2, pre-run
fraction (the soluble sub-fraction of bacterial cells expressing
the fusion protein); 3, flow through; 4, elution with 100 mM
imidazole; and 5-9, elution with 250 mM imidazole.
[0100] FIG. 6A-6C: Characterization of TAT-MTS-FRA highly purified
fusion proteins The four highly purified TAT-MTS-FRA fusion
proteins were characterized by a SDS-PAGE gel (FIG. 6A) and by
Western blot analyses using anti-His (FIG. 6B) or anti-frataxin
(FIG. 6C) antibodies.
[0101] Abbreviations: 1, TAT-MTSfra-FRA; 2, TAT-MTScs-FRA; 3,
TAT-TSlad-FRA; 4, TAT-MTSorf-FRA; and M, Marker.
[0102] FIG. 7A-7B: Internalization of TAT-MTSlad-FRA into cells and
their mitochondria FIG. 7A presents an immunoblot of a Western blot
analysis using anti-His antibodies and FIG. 7B presents an
immunoblot of a Western blot analysis using anti frataxin
antibodies performed with BJAB cells incubated in the absence
(lanes 1 & 2) or in the presence of TAT-MTSlad-FRA (lanes 3-7).
At the end of the incubation period, sub-fractionation was
performed, obtaining the cytoplasmic and mitochondrial fractions.
Fractions were separated by SDS-PAGE and subjected to Western blot
analysis.
[0103] Abbreviations: control, untreated cells: cytoplasm (1),
mitochondria (2); cells treated for 1 hr with the fusion protein:
cytoplasm (3), mitochondria (4); cells treated for 5 hr: cytoplasm
(5), mitochondria (6, 7; from two separate experiments); highly
purified TAT-MTSlad-FRA fusion protein as a positive control is
shown in (8). Arrow-heads indicate the fusion protein (or its
processing products, indicated by the lower arrow-head in FIG.
7B).
[0104] FIG. 8A-8B: Internalization of TAT-MTS-FRA fusion proteins
into mitochondria of cells FIG. 8A presents a Western blot analysis
using anti-frataxin antibodies of cells incubated for 3 hours with
TAT-MTS-FRA fusion proteins, each fusion protein at a final
concentration of 0.02 .mu.g/.mu.l. The cells were washed and their
mitochondria were isolated.
[0105] FIG. 8B presents a Western blot analysis using
anti-E1.alpha. antibodies of cells as detailed above.
Abbreviations: M, marker; mitochondria isolated from control cells
without any treatment (1), cells incubated with TAT-MTSfra-FRA (2),
cells incubated with TAT-MTScs-FRA (3), cells incubated with
TAT-MTSlad-FRA (4), cells incubated with TAT-MTSorf-FRA (5),
purified TAT-MTSfra-FRA fusion protein (6), and purified
TAT-MTScs-FRA fusion protein (7).
[0106] FIG. 9: Internalization of TAT-MTScs-FRA to fibroblasts of
FRA patients
[0107] An immunoblot of a Western blot analysis using an
anti-Frataxin antibody performed for mitochondrial (M) and
cytosolic (C) fractions of fibroblasts (F816) obtained from
Friedreich's ataxia patients that were incubated in the presence of
20 .mu.g/ml TAT-MTScs-FRA (or vehicle) for 2, 6 and 48 hours, with
fresh addition of TAT-MTScs-FRA (at 20 .mu.g/ml) after 24 hours.
TAT-MTScs-FRA and processed Frataxin are marked with arrows.
Abbreviations: kDa, kilodalton; TAT, transactivator of
transcription; MTS, mitochondria targeting sequence; CS, citrate
synthase; FXN, frataxin; Std, standard.
[0108] FIG. 10: Aconitase activity in fibroblasts obtained from
Friedreich's ataxia patients following 48 hours incubation with
TAT-MTScs-FRA
[0109] A bar graph showing aconitase activity (mOD/min) of
mitochondrial fractions obtained from Friedreich's ataxia patients'
fibroblasts (F816) incubated for 48 hours with either 20 .mu.g/ml
TAT-MTScs-FRA protein or vehicle (FXN or VEH, respectively). HepG2
whole cells homogenate served as positive control (POS.CON).
Abbreviations: kDa, kilodalton; TAT, transactivator of
transcription; MTS, mitochondria targeting sequence; CS, citrate
synthase; FXN, frataxin; Std, standard; VEH, vehicle, POS. CON.,
positive control.
[0110] FIG. 11A-11B: TAT-MTS-FRA fusion proteins partially rescue
cells from BSO-induced oxidative stress
[0111] FIG. 11A presents a bar diagram showing percentage of cell
death induced by L-Buthionine-sulfoximine (BSO). Normal lymphocytes
or lymphocytes obtained from Friedreich's ataxia (FRDA) patients
(Lym 43) were seeded, incubated for 5 hr with the various
TAT-MTS-FRA fusion proteins, after which BSO at different
concentrations was added for additional 48 hr. At the end of the
incubation time, cell cultures were subjected to cell proliferation
assays.
[0112] FIG. 11B presents a bar diagram showing percentage of cell
death of Lym 43 induced by L-Buthionine-sulfoximine (BSO)
correlated with caspase 3 activity within the cells, assessed using
the Apo-ONE Homogeneous Caspase 3/7 Assay Kit (Promega).
Experiments were carried in parallel with cell viability
assays.
[0113] Abbreviations: %, percent; YC, normal lymphocytes; FRA lym,
lymphocytes obtained from Friedreich's ataxia patients;
HTFRA=TAT-MTSfra-FRA, HT(LAD)FRA=TAT-MTSlad-FRA,
HT(ORF)FRA=TAT-MTSorf-FRA, HT(CS)FRA=TAT-MTScs-FRA; BSO,
L-Buthionine-sulfoximine; and PBS, Phosphate buffered saline.
[0114] FIG. 12A-12D: TAT-MTS-FRA fusion proteins partially rescue
fibroblasts obtained from patients from BSO-induced oxidative
stress, a comparison
[0115] FIG. 12A to FIG. 12D present bar diagrams of percentage of
cell death induced by BSO in fibroblasts obtained from FRDA
patients (Fib. 78). Cells were seeded, incubated for 24 hr with the
TAT-MTSfra-FRA (FIG. 12A), TAT-MTScs-FRA (FIG. 12B), TAT-MTSlad-FRA
(FIG. 12C) and with the TAT-MTSorf-FRA (FIG. 12D) fusion protein,
after which BSO at different concentrations was added for
additional 48 hr. At the end of the incubation time, cell cultures
were subjected to cell proliferation assays. Abbreviations: %,
percent; PBS, Phosphate buffered saline; BSO,
L-Buthionine-sulfoximine; HTF, TAT-MTSfra-FRA; MTS (cs),
TAT-MTScs-FRA; MTS (LAD), TAT-MTSlad-FRA; MTS (ORF),
TAT-MTSorf-FRA.
[0116] FIG. 13A-13B: TAT-MTSorf-FRA rescues lymphocytes and
fibroblasts obtained from Friedreich's ataxia patients from
BSO-induced oxidative stress
[0117] FIG. 13A presents a bar diagram of percentage of cell death
induced by BSO in fibroblasts obtained from FRDA patients (Fib. 78)
and FIG. 13B presents a bar diagram of percentage of cell death
induced by BSO in lymphocytes obtained from patients (Lym 43).
Cells were seeded, incubated for 24 hr with the TAT-MTSorf-FRA
fusion protein, after which BSO at different concentrations was
added for additional 48 hr. At the of the incubation time, cell
cultures were subjected to cell proliferation assays.
Abbreviations: PBS, Phosphate buffered saline; %, percent; BSO,
L-Buthionine-sulfoximine; and ORF, C6ORF66.
[0118] FIG. 14A-14B: TAT-MTSlad-FRA rescues fibroblasts obtained
from patients from BSO-induced oxidative stress
[0119] FIG. 14A and FIG. 14B present bar diagrams of percentage of
cell death induced by BSO in fibroblasts obtained from FRDA
patients (Fib. 78) of two representative experiments. Cells were
seeded, incubated for 24 hr with the TAT-MTSlad-FRA fusion protein,
after which BSO at different concentrations was added for
additional 48 hr. At the of the incubation time, cell cultures were
subjected to cell proliferation assays. Abbreviations: %, percent;
PBS, Phosphate buffered saline; BSO, L-Buthionine-sulfoximine; LAD,
lipoamide dehydrogenase.
[0120] FIG. 15A-15D: TAT-MTS-FRA fusion proteins constructs
[0121] FIG. 15A to FIG. 15D are schematic presentations of
TAT-MTS-FRA fusion protein constructs comprising a HIV-1
transactivator of transcription (TAT) domain (boxed) fused to a
GSDP linker (colored in grey) fused to a human mitochondria
targeting sequence (MTS) of a human mitochondrial protein
(double-underlined) selected from frataxin (MTSfra, FIG. 15A),
citrate synthase (MTScs, FIG. 15B), lipoamide dehydrogenase
(MTSlad, FIG. 15C) and C6ORF66 (MTSorf, FIG. 15D) fused to human
frataxin (underlined).
[0122] FIG. 16A-16E: Expression and purification of TAT-MTS-OTC
protein constructs
[0123] FIG. 16A to FIG. 16D are images of sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS PAGE) analysis of
bacterially expressed TAT-MTS-OTC fusion protein constructs
comprising ornithine transcarbamoylase (OTC, FIG. 16A), citrate
synthase (CS, FIG. 16B), C6ORF66 (ORF, FIG. 16C) or lipoamide
dehydrogenase (LAD FIG. 16D) as their MTS. The fusion protein
construct is indicated with arrows. About 2 .mu.g of each protein
and BSA were analyzed on 4-20% gels, followed by Coomassie blue
staining. Marker sizes are presented in FIG. 16E. Abbreviations:
M1, protein marker; kDa, kilodalton.
[0124] FIG. 17A-17B: Internalization of TAT-MTS-OTC protein
constructs into mitochondria FIG. 17A is an image of a Western blot
analysis performed for mitochondrial fractions of HepG2 cells
incubated with 12 .mu.g/ml of the TAT-MTS-OTC fusion protein
TAT-MTSotc-OTC, TAT-MTScs-OTC, TAT-MTSlad-OTC or vehicle for 20
min, 1 and 3 hours in either Eagle's Minimum Essential Medium
(EMEM) alone or EMEM supplemented with DMSO (D), Trehalose (T), or
Ornithine (O), as indicated. The protein standards TAT-MTSlad-OTC
or TAT-MTSorf-OTC (Std.) were run in parallel, sizes of the protein
markers (M) are presented in FIG. 17B. Migration of the OTC fusion
protein is marked by an arrow. Abbreviations: EMEM, Eagle's Minimum
Essential Medium; TAT, transactivator of transcription; MTS,
mitochondria targeting sequence; OTC, ornithine transcarbamoylase;
CS, citrate synthase; LAD, lipoamide dehydrogenase; Std., standard;
T/D, Trehalose/DMSO; T/D/O, Trehalose/DMSO/Ornithine; M,
marker.
[0125] FIG. 18A-18B: Western blot analysis of cytosolic fractions
of HepG2 cells incubated with TAT-MTS-OTC protein constructs
[0126] FIG. 18A and FIG. 18B are images of Western blot analyses
performed for cytosolic fractions of HepG2 cells incubated with 12
.mu.g/ml of the TAT-MTS-OTC fusion protein TAT-MTSotc-OTC,
TAT-MTScs-OTC, TAT-MTSlad-OTC or vehicle for 20 min, 1 and 3 hours
in either EMEM alone or EMEM supplemented with DMSO (D), Trehalose
(T), or Ornithine (0), as indicated. The protein standards
TAT-MTScs-OTC or TAT-MTSorf-OTC (Std.) were run in parallel.
Abbreviations: OTC, ornithine transcarbamoylase; CS, citrate
synthase; LAD, lipoamide dehydrogenase; ORF, C6ORF66; EMEM, Eagle's
Minimum Essential Medium; T/D, Trehalose/DMSO; T/D/O,
Trehalose/DMSO/Ornithine; M, marker.
[0127] FIG. 19: In vitro enzymatic activity of OTC
[0128] A bar graph showing the level of in vitro enzymatic
activities of the OTC protein constructs TAT-MTScs-OTC and
TAT-MTSotc-OTC measured by detecting the net absorbance of
citrulline at an optical density (O.D.) of 492 nm compared to a
control. Abbreviations: TAT, transactivator of transcription; MTS,
mitochondria targeting sequence; OTC, ornithine transcarbamoylase;
CS, citrate synthase.
[0129] FIG. 20: Rescue from ammonia stress by OTC fusion
proteins
[0130] A bar graph showing cell viability of HepG2 cells suffering
from ammonia stress in the presence of 14 .mu.g/ml of the OTC
protein constructs TAT-MTScs-OTC, and TAT-MTSotc-OTC, compared to
vehicle-treated and untreated cells. Florescence signal was
calculated as the ratio between Ammonium chloride treated and
non-treated cells. Abbreviations: TAT, transactivator of
transcription; MTS, mitochondria targeting sequence; CS, citrate
synthase; OTC, ornithine transcarbamoylase.
DETAILED DESCRIPTION OF EMBODIMENTS
[0131] The presently disclosed subject matter relates to the
preparation of various plasmid constructs encoding TAT-MTS-Frataxin
fusion proteins, providing a basis of a wide-range therapeutic tool
for delivering mitochondrial proteins into mitochondria.
[0132] The protein constructs described herein, comprising a
mitochondrial protein as well as TAT and a specific mitochondrial
targeting sequence (MTS), enabling the mitochondrial protein to
cross both cellular and mitochondrial membranes, were expressed and
purified and their biological activity was verified. Remarkably,
the protein yield obtained for fusion protein constructs comprising
an MTS which was other than the native MTS of the mitochondrial
protein present in the fusion construct (e.g., a frataxin fusion
protein construct with MTS heterologous to frataxin), was superior
to the yield obtained for a frataxin fusion protein construct
comprising the native MTS of frataxin.
[0133] As demonstrated below, the inventors show that various
fusion proteins are able to enter the mitochondria within intact
cells. In addition, the inventors show that the fusion proteins
exhibit biological activity. For example, the various TAT-MTS-FRA
fusion proteins were shown to rescue cells obtained from Friedreich
ataxia patients as well as normal cells from oxidative stress, as
demonstrated in the Examples below. Surprisingly, a superior
protective effect was observed for fusion proteins carrying an MTS,
which was other than the native MTS of the functional mitochondrial
protein present in the fusion construct (i.e. a heterologous MTS)
as compared to the effect demonstrated by the fusion protein
constructs carrying the native MTS.
[0134] The presently disclosed subject matter provides fusion
protein constructs comprising heterologous MTSs of human
nuclear-encoded mitochondrial proteins that are classical MTS
sequences, which are known to be removed upon entry to the
mitochondria. By a non-limiting example, a delivery system as
herein described comprising frataxin may be used for the treatment
or alleviation of Friedreich's ataxia or any other disorder
associated with a deficiency of frataxin or defective frataxin.
[0135] Thus, the presently disclosed subject matter provides a
fusion protein comprising a HIV-1 transactivator of transcription
(TAT) domain, a functional human mitochondrial protein and a human
mitochondria targeting sequence (MTS) situated between said TAT
domain and said functional human mitochondrial protein and wherein
said human MTS is heterologous to said functional human
mitochondrial protein.
[0136] The term "functional human mitochondrial protein" as used
herein refers to any protein which is essential for a biological
activity of mitochondria. A functional human mitochondrial protein
may be a protein, which is active when present in the mitochondria
by itself (per se) or a protein that when present in the
mitochondria functions as a component of a mitochondrial
multi-component complex (i.e. with other enzymes, co-factors, or
proteins). Typically, a functional human mitochondrial protein is a
protein, which, when absent, deficient or mutated, causes a
mitochondrial disorder or is associated with a mitochondrial
disorder.
[0137] In some specific embodiments, the functional mitochondrial
protein refers to the full-length amino acid sequence of the
protein. In other embodiments, the functional mitochondrial protein
is a fragment of the full-length amino acid sequence, sufficient to
provide the mitochondrial protein activity, either alone or as part
of a multi-component complex, as appropriate.
[0138] In further embodiments, the functional human mitochondrial
protein is a mutated derivative of said protein, wherein one or
more of the native amino acid residues has been deleted, replaced
or modified while still maintaining the mitochondrial functionally
of the protein (alone or as part of a multi-component complex).
[0139] In the above and other embodiments, the functional human
mitochondrial protein (also denoted "mature" protein) refers to a
protein devoid of its mitochondrial targeting sequence (MTS). In
other words, the fusion protein construct herein provided comprises
a functional mitochondrial protein, which, upon entry to the
mitochondria is cleaved off from the fusion protein construct in
its mature, active (functional) state.
[0140] By way of non-limiting example, in the above and other
embodiments of the disclosed subject matter, the functional human
mitochondrial protein whose activity is supplied by a fusion
protein of the present invention may be any one of human frataxin
(the mature protein having the amino acid sequence denoted by SEQ
ID NO. 26 and encoded by the nucleic acid sequence denoted by SEQ
ID NO. 6), ornithine transcarbamoylase (OTC, the mature protein
having the amino acid sequence denoted by SEQ ID NO. 39 and encoded
by the nucleic acid sequence denoted by SEQ ID NO. 15), human
Lipoamide Dehydrogenase (LAD), 2-oxoisovalerate dehydrogenase alpha
subunit (Branched-Chain Keto Acid Dehydrogenase E1a) (NCBI Protein
Database Accession No. P12694; OMIM:248600), 2-oxoisovalerate
dehydrogenase beta subunit (Branched-Chain Keto Acid Dehydrogenase
E1.beta.; P21953), Acyl-CoA dehydrogenase, medium-chain specific
(P1 1310; OMIM:201450), Acyl-CoA dehydrogenase, very-long-chain
specific (P49748; OMIM:201475), Trifunctional enzyme alpha subunit
(Long-chain 3 hydroxyacyl CoA Dehydrogenase or LCHAD) (P40939;
OMIM:609015) (HADHA), Trifunctional enzyme beta subunit
(Hydroxyacyl-CoA Dehydrogenase/3-Ketoacyl-CoA Thiolase/Enoyl-CoA
Hydratase (P55084) (HADHB)), Pyruvate dehydrogenase E1 component
beta subunit (P1 1177; OMIM:208800), and Pyruvate dehydrogenase E1
component alpha subunit (P08559; OMIM:312170).
[0141] In some embodiments, the human mitochondrial protein is a
functional mitochondrial protein per se and/or is a component of a
mitochondrial multi-component complex.
[0142] As indicated above, the functional human mitochondrial
protein of the disclosed subject matter may be a protein which is
active when present in the mitochondria by itself (i.e. the protein
per se is active) or a protein that when present in the
mitochondria functions as a component of a mitochondrial
multi-component complex (i.e. with other enzymes, co-factors, or
proteins). The term "mitochondrial multi-component complex" as used
herein refers to an enzyme that forms a complex with other enzymes
or proteins that is essential for a biological activity of
mitochondria.
[0143] As shown in the Examples below (FIG. 7), the fusion protein
comprising a TAT and MTS sequences is cleaved upon entry into the
mitochondria, and a mature active protein is obtained. The protein
constructs provided by the presently disclosed subject matter thus
allow a human mitochondrial protein, which is first covalently
attached to TAT and MTS domains, to cross both cellular and
mitochondrial membranes, and once inside the mitochondria, be
processed by mitochondrial peptidases while retaining their
biological activity and proper conformation. The delivery system
described herein thus enables a human mitochondrial protein to
retain its mitochondrial function per se or the integration thereof
in a mitochondrial multi-component complex.
[0144] The mitochondrial multi-component complex encompassed by the
present disclosure refers to a group of at least two different
proteins assembled together in a specific ratio that functions in a
coordinated fashion to catalyze a series of reactions. The function
of a mitochondrial multi-component complex is dependent on its
structure; thus, the proteins that compose the complex must
properly fold and physically fit together in the proper
configuration in order to efficiently catalyze the series of
reactions.
[0145] In all embodiments, the functional human mitochondrial
protein according to presently disclosed subject matter is cleaved
off from the fusion protein construct upon entry to the
mitochondria and resides therein at its mature, properly-folded
active state. In some embodiments, the functional human
mitochondrial protein may readily then integrate into a
conformationally-sensitive mitochondrial multi-component
complex.
[0146] By way of non-limiting example, the presently disclosed
subject matter encompasses a mitochondrial multi-component complex
which is any one of pyruvate dehydrogenase complex (PDHC),
.alpha.-ketoglutarate dehydrogenase complex (KGDHC), and
branched-chain keto-acid dehydrogenase complex (BCKDHC), the
complexes of the respiratory chain, and those involved in fatty
acid .beta.-oxidation and the urea cycle. The complexes of the
respiratory chain are complex I (NADH-ubiquinone oxidoreductase),
complex II (succinate-ubiquinone oxidoreductase), complex III
(ubiquinol-ferricytochrome C oxidoreductase), complex IV
(Cytochrome C oxidoreductase), and complex V (FIFO ATPase) where
each mitochondrial multicomponent complex represents a separate
embodiment of the present invention.
[0147] As shown in Examples 1-3 below, the inventors have cloned,
expressed and purified fusion protein constructs comprising the
protein frataxin.
[0148] The mitochondrial protein human frataxin (FXN) is an
essential and highly conserved protein expressed in most eukaryotic
organisms that appears to function in mitochondrial iron
homeostasis, notably the de novo biosynthesis of iron-sulfur
(Fe--S) cluster proteins and heme biosynthesis. The exact function
of FXN has not been defined but recent studies suggest that FXN
functions as an allosteric activator with Fe.sup.2+ for Fe--S
cluster biosynthesis. The absence of FXN is associated with a loss
of activity in Fe--S-containing proteins, such as aconitase as well
as with the disease Friedreich ataxia.
[0149] Precursor FXN protein (23.1 kDa, 210 amino acids) comprises
an 80 amino acid mitochondrial targeting sequence (MTS) at its
amino (N) terminus. Within mitochondria, the precursor FXN protein
is processed in two steps by the mitochondrial matrix processing
peptidase (MPP). It has been shown that the intermediate form of
FXN is formed by cleavage at residue 42 by the MPP, and the
resulting form of FXN (FXN42-210) has been shown to be cleaved at
amino acid 81, yielding a mature, 130 amino acid protein, with a
predicted molecular weight of 14.2 kDa.
[0150] As described above, Friedreich ataxia is an autosomal
recessive degenerative disorder characterized by ataxia, areflexia,
sensory loss, weakness, scoliosis, and cardiomyopathy. A deficiency
of frataxin in cells leads to decreased activities of mitochondrial
iron-sulfur cluster-containing enzymes, to an accumulation of iron
in the mitochondrial matrix, increased sensitivity to oxidative
stress, as well as to impaired adenosine triphosphate (ATP)
production.
[0151] In the above and other embodiments of the presently
disclosed subject matter, frataxin refers to human frataxin and any
biologically active fragments and derivatives thereof, which is
devoid of its natural (native) MTS sequence. Non limiting examples
for mature human frataxin are given by the accession number
Q16595[81-210] and as indicated in Table 1 below, where the amino
acid sequence of mature human frataxin is as set forth in SEQ ID
NO. 26 and the nucleic acid sequence encoding therefor is as set
forth in SEQ ID NO. 6.
[0152] Notably, as shown in Example 4 below, a TAT-MTS-frataxin
fusion protein was demonstrated by the inventors to enter
mitochondria of human intact BJAB cells. Analysis of sub-cellular
fractions of these cells, in order to separate the mitochondria
from the cytosol, verified that the various frataxin fusion protein
constructs (i.e. TAT-MTSlad-FRA, TAT-MTSfra-FRA, TAT-MTScs-FRA, and
TAT-MTSorf-FRA) were indeed successfully delivered into the
mitochondria. Surprisingly, among the fusion proteins carrying a
heterologous MTS, the MTS of citrate synthase (MTScs) was shown by
the inventors to be delivered into the mitochondria in the most
efficient manner.
[0153] Delivery of fusion proteins comprising a mitochondrial
protein into the mitochondria has far-reaching therapeutic
beneficial implications for treatment of mitochondrial disorders in
general, and delivery of frataxin into mitochondria has specific
therapeutic benefit for treatment of Friedreich's ataxia in
particular.
[0154] As noted above, ornithine transcarbamoylase (OTC, also
called ornithine carbamoyltransferase) is also encompassed by the
presently disclosed subject matter. OTC is a protein having
enzymatic activity that catalyzes the reaction between carbamoyl
phosphate (CP) and ornithine (Orn) to form citrulline (Cit) and
phosphate (Pt). In mammals OTC is located in the mitochondria and
is part of the urea cycle.
[0155] OTC is a trimer, and the active sites thereof are located at
the interface between the protein monomers, emphasizing the
importance of proper folding to the mitochondrial activity of the
protein. Deficiency in OTC results in an increase in ammonia level,
leading to neurological problems.
[0156] As demonstrated in the appended Examples, fusion protein
constructs according to the invention comprising OTC and its native
MTS as well as fusion protein constructs comprising OTC and a
heterologous MTS were able to internalize into the mitochondria in
HepG2 cells (Example 9). Interestingly, the internalization ability
of the fusion protein constructs comprising an MTS that is
heterologous to OTC was slightly higher than the internalization
ability of the fusion protein construct comprising the native MTS
of OTC. Fusion protein constructs comprising the protein OTC were
also shown to be active. As shown in FIG. 20, the level of cell
viability in the presence of fusion protein constructs comprising
OTC and citrate synthase as the MTS was similar to the level of
cell viability for cells which were not exposed to ammonium
chloride (which served as a model for ammonia stress conferred by
defective or missing OTC).
[0157] Furthermore, the level of cell viability in the presence of
a fusion protein construct comprising OTC and citrate synthase as
the MTS was higher from the level of cell viability in cells
treated with the fusion protein construct comprising the native MTS
of OTC.
[0158] Thus, in the above and other embodiments of the disclosed
subject matter, the functional human mitochondrial protein is
specifically any one of frataxin and ornithine transcarbamoylase
(OTC). In some embodiments, the nucleic acid encoding the mature
OTC protein is denoted by SEQ ID NO. 15. In other embodiments the
OTC mature protein of the present disclosure has the amino acid
sequence denoted by SEQ ID NO. 39.
[0159] As indicated above, the presently disclosed subject matter
provides a fusion protein comprising a HIV-1 transactivator of
transcription (TAT) domain, a functional human mitochondrial
protein and a human mitochondria targeting sequence (MTS) situated
between said TAT domain and said functional mitochondrial protein
and wherein said human MTS is heterologous to said functional
protein.
[0160] Most of the proteins directed to the mitochondria are
synthesized with a mitochondrial targeting (or translocation)
sequence (MTS), which allows their import from the cytoplasm into
mitochondria through the translocation machinery. Once entering the
mitochondria, the MTS is recognized and cleaved off, allowing for
proper processing and, if necessary, assembly into mitochondrial
enzymatic complexes.
[0161] Thus, as used herein, the term "mitochondria targeting
sequence", MTS or "mitochondria translocation sequence" refers to
an amino acid sequence capable of causing the transport into the
mitochondria of a protein, peptide, amino acid sequence, or
compound attached thereto, and any biologically active fragments
thereof. MTSs used in the fusion protein constructs in accordance
with the presently disclosed subject matter, which are situated
N-terminal to the functional mitochondrial protein, are typically
from about 15 to about 40 amino acids in length, including from
about 3 to about 5 nonconsecutive basic amino acid
(arginine/lysine) residues, often with several serine/threonine
residues but without acidic amino acid (asparate/glutamate)
residues. In their molecular structure, these MTS s are able to
form strong basic amphipathic .alpha.-helices that are essential
for efficient mitochondrial transportation.
[0162] In other words, presently disclosed is a fusion protein as
herein defined, wherein said functional human mitochondrial protein
is C-terminal to said human mitochondria targeting sequence
(MTS).
[0163] The term "about" as used herein indicates values that may
deviate up to 1%, more specifically 5%, more specifically 10%, more
specifically 15%, and in some cases up to 20% higher or lower than
the value referred to, the deviation range including integer
values, and, if applicable, non-integer values as well,
constituting a continuous range.
[0164] In the above and other embodiments, the MTS is human MTS,
namely MTS of a human mitochondrial protein.
[0165] In the above and other embodiments of the presently
disclosed subject matter the MTS comprises from about 15 to about
40 amino acid residues, including from about 3 to about 5
nonconsecutive (i.e. which are not covalently linked one to the
other in a sequential manner) basic amino acid residues, and
optionally from about 1 to about 3 or 4 or 5 serine/threonine
residues.
[0166] The term "amino acid residues" as used herein refers to
naturally occurring and synthetic amino acids, as well as amino
acid analogs and amino acid mimetics that can function in a manner
similar to the naturally occurring amino acids. Naturally occurring
amino acids are those encoded by the genetic code, as well as those
amino acids that are later modified, e.g., hydroxyproline,
.gamma.-carboxyglutamate, and O-phosphoserine. "amino acid analogs
and amino acid mimetics" refers to compounds that have the same
fundamental chemical structure as a naturally occurring amino acid.
Such analogs have modified R groups or modified peptide backbones,
but retain the same basic chemical structure as a naturally
occurring amino acid. Amino acids may be referred to herein by
either their commonly known three letter symbols or by the
one-letter symbols recommended by the IUPAC-IUB Biochemical
Nomenclature Commission.
[0167] It is well known in the art that amino acid residues may be
divided according to their chemical properties to various groups,
inter alia, on the basis of similarity in polarity, charge,
solubility, hydrophobicity, hydrophilicity, and/or the amphipathic
nature of the residues involved.
[0168] For example, nonpolar "hydrophobic" amino acids are selected
from the group consisting of Valine (V), Isoleucine (I), Leucine
(L), Methionine (M), Phenylalanine (F), Tryptophan (W), Cysteine
(C), Alanine (A), Tyrosine (Y), Histidine (H), Threonine (T),
Serine (S), Proline (P), Glycine (G), Arginine (R) and Lysine (K);
"polar" amino acids are selected from the group consisting of
Arginine (R), Lysine (K), Aspartic acid (D), Glutamic acid (E),
Asparagine (N), Glutamine (Q); "positively charged" amino acids are
selected form the group consisting of Arginine (R), Lysine (K) and
Histidine (H) and wherein "acidic" amino acids are selected from
the group consisting of Aspartic acid (D), Asparagine (N), Glutamic
acid (E) and Glutamine (Q). "Basic" amino acids are selected from
the group consisting of Histidine (H), lysine (K) and Arginine (R),
which are polar and positively charged at pH values below their
pKa's, and are very hydrophilic.
[0169] As indicated above, the presently disclosed subject matter
encompasses human mature frataxin and any biologically active
fragments and derivatives thereof, which is devoid of its natural
(native) MTS sequence. By the term "biologically active fragments
and derivatives" it is meant any variations, including deletion,
substitution and/or insertion of one or more amino acid residues in
the amino acid sequences of mature frataxin (or in the nucleic acid
encoding therefor), for example 1, 2, 3, 4, 5 or more amino acid
residues, in accordance with the presently disclosed subject matter
which would not alter the biological activity of frataxin.
[0170] The invention further relates to DNA constructs comprising
the nucleic acid sequence of the presently disclosed subject matter
or biologically functional fragments and derivatives thereof. The
constructs of the presently disclosed subject matter may further
comprise additional elements such as promoters, regulatory and
control elements, translation, expression and other signals,
operably linked to the nucleic acid sequence of the invention.
[0171] It is known that each mitochondrial enzyme produced in the
cytoplasm and transported into the mitochondria is produced as a
precursor protein, carrying its natural MTS. Thus, the precursor
mitochondrial protein already has its native MTS. However, this
naturally occurring sequence in the precursor protein may be
exchanged with any other known MTS.
[0172] As exemplified herein, the fusion protein constructs
comprising frataxin or ornithine transcarbamoylase prepared by the
inventors further comprised the MTS of lipoamide dehydrogenase
(MTSlad, of the amino acid sequence denoted by SEQ ID NO. 24 and
encoded by the nucleic acid sequence denoted by SEQ ID NO. 5),
C6ORF66 (MTSorf, of the amino acid sequence denoted by SEQ ID NO.
25 and encoded by the nucleic acid sequence denoted by SEQ ID NO.
4), or of citrate synthase (MTScs, of the amino acid sequence
denoted by SEQ ID NO. 23 and encoded by the nucleic acid sequence
denoted by SEQ ID NO. 3).
[0173] Alternatively, fusion protein constructs comprising frataxin
or ornithine transcarbamoylase prepared by the inventors comprised
respectively the native MTS of frataxin (MTSfra, of the amino acid
sequence denoted by SEQ ID NO. 22 and encoded by the nucleic acid
sequence denoted by SEQ ID NO. 2) or the native MTS of ornithine
transcarbamoylase (MTSotc, of the amino acid sequence denoted by
SEQ ID NO. 38 and encoded by the nucleic acid sequence denoted by
SEQ ID NO. 37).
[0174] In addition, the inventors showed that fusion protein
constructs comprising frataxin and a MTS sequence that is not the
native MTS of frataxin (i.e. heterologous MTS) were superior to the
frataxin fusion protein construct comprising the native MTS, based
on the higher yield obtained for fusion protein constructs
comprising heterologous MTS during the expression and purification
stages.
[0175] Surprisingly, fusion protein constructs comprising frataxin
and a heterologous MTS were also demonstrated by the inventors to
have an enhanced biological activity as compared to the frataxin
fusion protein construct comprising the native MTS (Example 5). In
particular, a fusion protein construct comprising frataxin and
citrate synthase MTS showed the highest effect in reducing toxicity
of BSO (FIG. 11A and FIG. 11B). As shown in Example 4 below, the
fusion protein construct comprising frataxin and citrate synthase
MTS also showed the highest ability of being delivered into
mitochondria among the exemplified constructs comprising
heterologous MTS.
[0176] Consistent with the above results, a fusion protein
construct comprising frataxin and another heterologous MTS, namely
the MTS of lipoamide dehydrogenase (LAD), was also demonstrated by
the inventors to have an enhanced biological activity as compared
to the frataxin fusion protein construct comprising the frataxin
native MTS, as demonstrated in FIG. 12C. As evident from FIG. 12,
the biological activity of this fusion protein construct was
comparable to the biological activity of the fusion protein
construct comprising the citrate synthase MTS.
[0177] As detailed above, it is known that FXN mRNA is translated
into a precursor polypeptide that is transported to the
mitochondrial matrix and processed to at least two forms, namely
FXN42-210 and FXN81-210, where FXN42-210 is the transient
processing intermediate and FXN81-210 represents the mature
protein. In other words, the transient frataxin polypeptide
FXN42-210 includes a portion of the native frataxin MTS, whereas
the FXN81-210 is the mature protein per se, devoid of its native
MTS. Without wishing to be bound by theory, by using a heterologous
MTS in fusion protein constructs comprising frataxin, mature
frataxin is expected to be released from the fusion protein at a
single step, thereby raising the biological availability of this
protein in the mitochondria compared to fusion protein constructs
comprising frataxin and its native MTS.
[0178] Thus, the MTS encompassed by the presently disclosed subject
matter is any human MTS that is encoded by the nuclear DNA,
translated (produced) in the cytoplasm and transported into the
mitochondria and which is not the native N-terminal MTS sequence of
the functional protein present in the fusion protein construct
according to the invention. In other words, the MTS sequence is
other than the native N-terminal MTS sequence of the functional
protein, i.e. is heterologous thereto. The various MTS may be
exchangeable for each mitochondrial enzyme among themselves. Each
possibility represents a separate embodiment of the present
invention.
[0179] As used herein, the term "heterologous" when referring to
MTS fused to the functional human mitochondrial protein according
to the present disclosure, is to be taken to mean MTS obtained from
another (distinct) mitochondrial protein, i.e. MTS which is not the
naturally occurring MTS of the said functional protein.
[0180] By way of a non-binding example, the heterologous MTS
according to the present disclosure is a MTS which is heterologous
to the mitochondrial protein frataxin, or to the mitochondrial
protein ornithine transcarbamoylase (OTC) as exemplified herein,
which may be, but is not limited to, the MTS of any human
mitochondrial protein, e.g. lipoamide dehydrogenase (MTSlad, of the
amino acid sequence denoted by SEQ ID NO. 24 and encoded by the
nucleic acid sequence denoted by SEQ ID NO. 5), C6ORF66 (MTSorf, of
the amino acid sequence denoted by SEQ ID NO. 25 and encoded by the
nucleic acid sequence denoted by SEQ ID NO. 4), and of citrate
synthase (MTScs, of the amino acid sequence denoted by SEQ ID NO.
23 and encoded by the nucleic acid sequence denoted by SEQ ID NO.
3), which are non-limiting examples.
[0181] Thus, in embodiments of the presently disclosed fusion
protein constructs, the MTS can be any one of human mitochondrial
citrate synthase MTS having the amino acid sequence denoted by SEQ
ID NO. 23, the human lipoamide dehydrogenase MTS having the amino
acid sequence denoted by SEQ ID NO. 24, the MTS of the human
C6ORF66 gene product having the amino acid sequence denoted by SEQ
ID NO. 25 and the MTS of human mitochondrial GLUD2 encoded by the
nucleic acid sequence denoted by SEQ ID NO. 16.
[0182] In other embodiments of the presently disclosed fusion
protein construct, the MTS is any one of human mitochondrial
citrate synthase MTS having the amino acid sequence denoted by SEQ
ID NO. 23 and the human lipoamide dehydrogenase MTS having the
amino acid sequence denoted by SEQ ID NO. 24.
[0183] In some embodiments disclosed is a fusion protein comprising
a HIV-1 transactivator of transcription (TAT) domain fused to human
frataxin and a human mitochondria targeting sequence (MTS) of a
human mitochondrial protein selected from lipoamide dehydrogenase
(LAD) and citrate synthase (CS) situated between said TAT domain
and said frataxin, wherein said frataxin is C-terminal to said MTS
of human lipoamide dehydrogenase or human citrate synthase.
[0184] In other embodiments disclosed is a fusion protein
comprising a HIV-1 transactivator of transcription (TAT) domain
fused to human ornithine transcarbamoylase (OTC) and a human
mitochondria targeting sequence (MTS) of a human mitochondrial
protein selected from lipoamide dehydrogenase (LAD) and citrate
synthase (CS) situated between said TAT domain and said OTC,
wherein said OTC is C-terminal to said MTS of human lipoamide
dehydrogenase or human citrate synthase.
[0185] As indicated above, the fusion protein according to the
presently disclosed subject matter comprises a HIV-1 transactivator
of transcription (TAT) domain, located at the N-terminus of the
fusion protein, N-terminal to the MTS as defined above, which in
turn is situated N-terminal to the functional human mitochondrial
protein (see FIG. 1 for a schematic presentation).
[0186] As used herein, the term HIV-1 transactivator of
transcription (TAT) domain refers to a protein transduction domain
which is an 11-amino-acid (residues 47-57) arginine- and
lysine-rich portion of the MV-I Tat protein having the amino acid
sequence YGRKKRRQRRR as set forth in SEQ ID NO. 21. TAT-fusion
protein constructs are known in the art to be introduced into
cultured cells, intact tissue, and live tissues and cross the
blood-brain barrier (BBB). TAT fusion proteins are also known to
traverse mitochondrial membranes [13].
[0187] The presently disclosed subject matter also encompasses any
fragments of the above defined TAT domain. For example, a TAT
domain according to the presently disclosed subject matter may
comprise from about 3 to about 11 (e.g. 4-11, 5-11, 6-11, 7-11,
8-11, 9, 10 or 11) sequential amino acid residues of the HIV-I Tat
protein having the amino acid sequence YGRKKRRQRRR (SEQ ID NO.
21).
[0188] In some embodiments, the fragment of the above defined TAT
domain comprise 9 sequential amino acid residues of the HIV-I Tat
protein, having the amino acid sequence of RKKRRQRRR, as set forth
in SEQ ID NO. 27 and encoded by the nucleic acid sequence denoted
by SEQ ID NO. 1, which was used in the preparation of the fusion
protein constructs exemplified below.
[0189] Thus, in this and other embodiments of the presently
disclosed subject matter, the fusion protein comprises a TAT domain
at its N-terminus and a functional mitochondrial protein at its
C-terminus, both covalently linked (fused) to an MTS that is
situated between said TAT domain and said functional mitochondrial
protein. In other words, the disclosure provides a protein
construct comprising an N-terminal TAT fused to N-terminal of MTS
fused to N-terminal of functional protein, as schematically
presented in FIG. 1 for frataxin.
[0190] The fusion protein according to the presently disclosed
subject matter may be prepared by any method known to a skilled
artisan. By example, the fusion protein as herein defined may be
prepared as exemplified below, by standard molecular biology and
cloning techniques.
[0191] The term "fusion protein" in the context of the invention
concerns a sequence of amino acids, predominantly (but not
necessarily) connected to each other by peptidic bonds. The term
"fused" in accordance with the fusion protein of the invention
refers to the fact that the amino acid sequences of at least three
different origins, namely, the TAT domain, the sequence of the
heterologous mitochondrial targeting domain (MTS) and the
functional mitochondrial protein, are linked to each other by
covalent bonds either directly or via an amino acid linker joining
(bridging, conjugating, covalently binding) the amino acid
sequences. The fusion may be by chemical conjugation such as by
using state of the art methodologies used for conjugating
peptides.
[0192] The fusion protein in the context of the invention may also
optionally cc p se at least one linker covalently joining different
domains of the fusion protein construct.
[0193] The term "linker" in the context of the invention concerns
an amino acid sequence of from about 4 to about 20 amino acid
residues positioned between the different fusion protein domains
and covalently joining them together. For example, a linker in
accordance with the invention may be 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19 or 20 amino acid long. Linkers are often
composed of flexible amino acid residues, for example but not
limited to glycine and serine so that the adjacent protein domains
are free to move relative to one another. The term "linker" can be
interchageably used with "spacer".
[0194] The design of a linker that enables proper folding of the
various domains of a protein is well known in the art. A
non-binding example of a linker is the amino acid sequence GSDP
(Gly-Ser-Asp-Pro) as denoted by SEQ ID NO. 32, which was used in
the Examples below to construct the fusion proteins His TAT MTS(cs)
81-210 FRA (denoted by SEQ ID NO. 17), His TAT MTS(fra) 81-210 FRA
(denoted by SEQ ID NO. 18), His TAT MTS(lad) 81-210 FRA (denoted by
SEQ ID NO. 19) and His TAT MTS(orf) 81-210 FRA (denoted by SEQ ID
NO. 20) as well as several of the fusion proteins comprising
OTC.
[0195] Thus in some embodiments the present disclosure relates to a
fusion protein as herein defined further comprising a linker
covalently linking said TAT domain to said MTS sequence.
[0196] The fusion protein in the context of the invention may also
optionally comprise at least one methionine (M) residue at its
N-terminus, as in the case of the exemplified fusion proteins
below. The methionine is positioned N-terminal to the TAT
domain.
[0197] Fusion may also be achieved by recombinant techniques, i.e.
by construction of a nucleic acid. sequence coding for the entire
the fusion protein (coding for all segments) so that essentially
all the bonds are peptidic bonds.
[0198] In order to facilitate purification of the protein
constructs described herein, fusion protein constructs in
accordance with the invention may also comprise an N-terminal tag
(e.g. His tag as exemplified below, Glutathione S-transferase
(GST), Maltose-Binding Protein (MBP), FLAG octapeptide, to name but
few), which may be removed or retained in the final fusion
construct. Such tags are normally cleaved off from the fusion
protein upon entry to the mitochondria, along with the TAT and MTS
sequences.
[0199] In some embodiments, the amino acid sequence of a fusion
protein according to the invention is as set forth in SEQ ID NO.
17, namely His TAT MTS(cs) FRA, SEQ ID NO. 19, namely His TAT
MTS(lad) FRA, SEQ ID NO. 20, namely, His TAT MTS(orf) FRA, SEQ ID
NO. 45, namely, His TAT MTS(cs) OTC, SEQ ID NO. 46, namely, His TAT
MTS(orf) OTC as well as SEQ ID NO. 47, namely, His TAT MTS(lad)
OTC.
[0200] Fusion protein constructs in accordance with the invention
may also be prepared without an N-terminal tag. In some
embodiments, the amino acid sequence of a fusion protein according
to the invention is as set forth in SEQ ID NO. 28, namely TAT
MTS(cs) FRA, SEQ ID NO. 30, namely TAT MTS(lad) FRA, SEQ ID NO. 31,
namely, TAT MTS(orf) FRA, SEQ ID NO. 49, namely TAT MTS(cs) OTC,
SEQ ID NO. 50, namely TAT MTS(orf) OTC, as well as in SEQ ID NO.
51, namely TAT MTS(lad) OTC.
[0201] Therefore the present disclosure further encompasses a
fusion protein having the amino acid sequence denoted by SEQ ID NO.
30, comprising a HIV-1 transactivator of transcription (TAT) domain
having the amino acid sequence denoted by SEQ ID NO. 27 fused to
human frataxin having the amino acid sequence denoted by SEQ ID NO.
26 and a mitochondria targeting sequence (MTS) of human lipoamide
dehydrogenase having the amino acid sequence denoted by SEQ ID NO.
24, said MTS situated between said TAT domain and said frataxin and
is linked to said TAT domain via a linker having the amino acid
sequence denoted by SEQ ID NO. 32, and wherein said frataxin is
C-terminal to said MTS of human lipoamide dehydrogenase.
[0202] In some embodiments the fusion protein as herein defined has
the amino acid sequence denoted by SEQ ID NO. 28, comprising a
HIV-1 transactivator of transcription (TAT) domain having the amino
acid sequence denoted by SEQ ID NO. 27 fused to human frataxin
having the amino acid sequence denoted by SEQ ID NO. 26 and a
mitochondria targeting sequence (MTS) of human citrate synthase
having the amino acid sequence denoted by SEQ ID NO. 23, said MTS
situated between said TAT domain and said frataxin, and is linked
to said TAT domain via a linker having the amino acid sequence
denoted by SEQ ID NO. 32, and wherein said frataxin is C-terminal
to said MTS of human citrate synthase.
[0203] In some embodiments the fusion protein as herein defined has
the amino acid sequence denoted by SEQ ID NO. 45, comprising a
HIV-1 transactivator of transcription (TAT) domain having the amino
acid sequence denoted by SEQ ID NO. 27 fused to human ornithine
transcarbamoylase (OTC) having the amino acid sequence denoted by
SEQ ID NO. 39 and a mitochondria targeting sequence (MTS) of human
citrate synthase having the amino acid sequence denoted by SEQ ID
NO. 23, said MTS situated between said TAT domain and said OTC, and
is linked to said TAT domain via a linker having the amino acid
sequence denoted by SEQ ID NO. 32, and wherein said OTC is
C-terminal to said MTS of human citrate synthase.
[0204] The presently disclosed subject matter further provides a
composition comprising a physiologically acceptable carrier and as
an active ingredient a fusion protein as herein defined.
[0205] In specific embodiments the presently disclosed subject
matter provides a composition comprising as an active ingredient a
fusion protein having the amino acid sequence denoted by SEQ ID NO.
45, denoted by SEQ ID NO. 30, or having the amino acid sequence
denoted by SEQ ID NO. 28 and a physiologically acceptable
carrier.
[0206] Also provided by the presently disclosed subject matter is a
pharmaceutical composition comprising a pharmaceutically acceptable
carrier and as an active ingredient a fusion protein construct as
herein defined.
[0207] The "composition" as herein defined generally comprises a
buffering agent, an agent which adjusts the osmolarity thereof, and
optionally, one or more pharmaceutically (or physiologically)
acceptable carriers, diluents, additives and excipients as known in
the art. Supplementary active ingredients can also be incorporated
into the compositions. The pharmaceutically acceptable carrier can
be 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 vegetable oils. Each carrier should be physiologically
or pharmaceutically acceptable, as the case may be, in the sense of
being compatible with the other ingredients and not injurious to
the patient.
[0208] The additives may be but are not limited to at least one of
a protease inhibitor, for example phenylmethanesulfonylfluoride or
phenylmethylsulfonyl fluoride (PMSF), Nafamostat Mesylate,
4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF),
Bestatin, Pepstatin A, E-64, Leupeptin, 1, 10-Phenanthroline and
any other protease inhibitor known in the art.
[0209] The "pharmaceutical compositions" of the presently disclosed
subject matter are compositions as described above, comprising
pharmaceutically acceptable carriers, diluent, adjuvant and/or
excipients and/or additives as known in the art.
[0210] The presently disclosed subject matter further provides a
pharmaceutical composition as herein defined for treating or
alleviating a mitochondrial disorder.
[0211] The term "mitochondrial disorder" as encompassed by the
presently disclosed subject matter refers to a group of systemic
diseases caused by inherited or acquired damage to the mitochondria
causing an energy shortage within those areas of the body that
consume large amounts of energy such as the liver, muscles, brain,
and the heart. The result is often liver failure, muscle weakness,
fatigue, and problems with the heart, eyes, and various other
systems.
[0212] The mitochondrial disorder may be any one of frataxin
deficiency which causes or is associated with Friedreich's ataxia;
a deficiency in OTC (X-linked recessive genetic disorder caused by
non-conservative mutations in the OTC gene); disorder associated
with LAD deficiency; or the mitochondrial metabolic disorder is
Complex I deficiency (OMIM:252010). Complex I deficiency can be
caused by a mutation in any of the subunits thereof. Alternatively,
the Complex I deficiency is caused by a mutation in a gene selected
from NDUFV1 (OMIM: 161015), NDUFV2 (OMIM:600532), NDUFS1 (OMIM:
157655), NDUFS2 (OMIM:602985), NDUFS3 (OMIM:603846), NDUFS4
(OMIM:602694), NDUFS6 (OMIM:603848), NDUFS7 (OMIM:601825), NDUFS8
(OMIM:602141), and NDUF A2 (OMIM: 602137).
[0213] In other embodiments, the mitochondrial disorder is Complex
IV deficiency (Cytochrome C oxidase; OMIM:220110). Complex IV
deficiency can be caused by a mutation in any of the subunits
thereof. In another embodiment, the Complex IV deficiency is caused
by a mutation in a gene selected from the group consisting of MTCO1
(0MIM:516030), MTCO2 (0MIM:516040), MTCO3 (0MIM:516050), COX1O
(0MIM:602125), COX6B1 (OMIM: 124089), SCO1 (OMIM:603644), FASTKD2
(0MIM:612322), and SC02 (OMIM:604272).
[0214] In other embodiments, the mitochondrial disorder is a
neurodegenerative disease. As provided herein, compositions of the
present invention exhibit the ability to traverse the blood-brain
barrier (BBB).
[0215] In further embodiments of the presently disclosed subject
matter, the mitochondrial disorder is selected from the group
consisting of encephalopathy and liver failure that is accompanied
by stormy lactic acidosis, hyperammonemia and coagulopathy. In
other embodiments, the mitochondrial disorder is selected from the
group consisting of Ornithine Transcarbamoylase deficiency
(hyperammonemia) (OTCD), Carnitine O-palmitoyltransferase II
deficiency (CPT2), Fumarase deficiency, Cytochrome c oxidase
deficiency associated with Leigh syndrome, Maple Syrup Urine
Disease (MSUD), Medium-Chain Acyl-CoA Dehydrogenase deficiency
(MCAD), Acyl-CoA Dehydrogenase Very Long-Chain deficiency (LCAD),
Trifunctional Protein deficiency, Progressive External
Ophthalmoplegia with Mitochondrial DNA Deletions (POLG), DGUOK,
TK2, Pyruvate Decarboxylase deficiency, and Leigh Syndrome (LS). In
another embodiment, the mitochondrial metabolic disorder is
selected from the group consisting of Alpers Disease; Barth
syndrome; beta-oxidation defects; carnitine-acyl-carnitine
deficiency; carnitine deficiency; co-enzyme Q10 deficiency; Complex
II deficiency (OMIM:252011), Complex III deficiency (OMIM: 124000),
Complex V deficiency (OMIM:604273), LHON-Leber Hereditary Optic
Neuropathy; MM--Mitochondrial Myopathy; LIMM-Lethal Infantile
Mitochondrial Myopathy; MMC--Maternal Myopathy and Cardiomyopathy;
NARP-Neurogenic muscle weakness, Ataxia, and Retinitis Pigmentosa;
Leigh Disease; FICP-Fatal Infantile Cardiomyopathy Plus, a
MELAS-associated cardiomyopathy; MELAS-Mitochondrial
Encephalomyopathy with Lactic Acidosis and Strokelike episodes;
LDYT-Leber's hereditary optic neuropathy and Dystonia;
MERRF-Myoclonic Epilepsy and Ragged Red Muscle Fibers;
MHCM-Maternally inherited Hypertrophic CardioMyopathy; CPEO-Chronic
Progressive External Ophthalmoplegia; KSS-Kearns Sayre Syndrome;
DM-Diabetes Mellitus; DMDF Diabetes Mellitus+Deafness; CIPO-Chronic
Intestinal Pseudoobstruction with myopathy and Ophthalmoplegia;
DEAF-Maternally inherited DEAFness or aminoglycoside-induced
DEAFness; PEM-Progres sive encephalopathy; SNHL-SensoriNeural
Hearing Loss; Encephalomyopathy; Mitochondrial cytopathy; Dilated
Cardiomyopathy; GER-Gastro intestinal Reflux; DEMCHO-Dementia and
Chorea; AMDF-Ataxia, Myoclonus; Exercise Intolerance; ESOC
Epilepsy, Strokes, Optic atrophy, and Cognitive decline; FBSN
Familial Bilateral Striatal Necrosis; FSGS Focal Segmental
Glomerulosclerosis; LIMM Lethal Infantile Mitochondrial Myopathy;
MDM Myopathy and Diabetes Mellitus; MEPR Myoclonic Epilepsy and
Psychomotor Regression; MERME MERRF/MELAS overlap disease; MHCM
Maternally Inherited Hypertrophic CardioMyopathy; MICM Maternally
Inherited Cardiomyopathy; MILS Maternally Inherited Leigh Syndrome;
Mitochondrial Encephalocardio myopathy; Multisystem Mitochondrial
Disorder (myopathy, encephalopathy, blindness, hearing loss,
peripheral neuropathy); NAION Nonarteritic Anterior Ischemic Optic
Neuropathy; NIDDM Non-Insulin Dependent Diabetes Mellitus; PEM
Progressive Encephalopathy; PME Progressive Myoclonus Epilepsy; RTT
Rett Syndrome; SIDS Sudden Infant Death Syndrome; MIDD Maternally
Inherited Diabetes and Deafness; and MODY Maturity-Onset Diabetes
of the Young, and MNGIE.
[0216] Each mitochondrial disease represents an embodiment of the
present invention.
[0217] In the above and other embodiments, the presently disclosed
subject matter provides a pharmaceutical composition as herein
defined for use in the treatment of Friedreich's ataxia or any
other disorder associated with deficiency of frataxin or defective
frataxin or for use in the treatment of a disorder associated with
a deficiency of OTC or with defective OTC.
[0218] In still further embodiments, the presently disclosed
subject matter provides a pharmaceutical composition for the
treatment of Friedreich's Ataxia by intravenous administration to a
subject in need thereof, said composition comprising a
therapeutically effective amount of a fusion protein as herein
defined.
[0219] In specific embodiments the presently disclosed subject
matter provides a pharmaceutical composition for the treatment of
Friedreich's Ataxia by intravenous administration to a subject in
need thereof, said composition comprising a therapeutically
effective amount of a fusion protein having the amino acid sequence
denoted by SEQ ID NO. 30 or having the amino acid sequence denoted
by SEQ ID NO. 28.
[0220] In further specific embodiments the presently disclosed
subject matter provides a pharmaceutical composition for the
treatment of Friedreich's Ataxia by intravenous administration to a
subject in need thereof, said composition comprising a
therapeutically effective amount of a fusion protein having the
amino acid sequence denoted by SEQ ID NO. 30.
[0221] In still further specific embodiments the presently
disclosed subject matter provides a pharmaceutical composition for
the treatment of Friedreich's Ataxia by intravenous administration
to a subject in need thereof, said composition comprising a
therapeutically effective amount of a fusion protein having the
amino acid sequence denoted by SEQ ID NO. 28.
[0222] The presently disclosed subject matter further provides a
fusion protein according to the invention for use in a method for
the treatment of a mitochondrial disorder.
[0223] In the above and other embodiments of the presently
disclosed subject matter the functional human mitochondrial protein
is frataxin for use in a method for the treatment or alleviation of
Friedreich's ataxia or any other disorder associated with
deficiency of frataxin or defective frataxin.
[0224] In the above and other embodiments of the presently
disclosed subject matter the functional human mitochondrial protein
is OTC for use in a method for the treatment or alleviation of a
disorder associated with a deficiency of OTC or with defective
OTC.
[0225] As shown below in Example 7, the inventors have shown that
fusion proteins comprising TAT, MTS and frataxin (TAT-MTS-FRA) were
able to partially rescue cells obtained from Friedreich's ataxia
patients, as well as normal cells, from oxidative stress,
exhibiting a clear biological activity of the fusion protein
constructs of the presently disclosed subject matter.
[0226] L-Buthionine sulphoximine (BSO) is an inhibitor of
gamma-glutamylcysteine synthetase (gamma-GCS) and, consequently
lowers tissue glutathione (GSH) concentrations. GSH plays an
important role in cellular defense against a wide variety of toxic
electrophiles via the formation of thioether conjugates. Therefore,
BSO was used by the inventors to model oxidative stress, through
its ability to inhibit de novo glutathione synthesis, thereby
depleting an important component of these cells' intrinsic defenses
against reactive oxygen species (ROS) and allowing for the
accumulation of ROS produced by natural cell processes, known to
result in cell death.
[0227] It is known that Friedreich ataxia cells are extremely
sensitive to BSO-induced oxidative stress compared with normal
cells because they lack Frataxin, and thus are used as an in vitro
model of the long-term consequences of absent Frataxin.
[0228] As shown in the Examples below, oxidative stress was induced
with various concentrations of BSO in cells obtained from patients
as well as in normal healthy cells and the effect of the various
TAT-MTS-FRA fusion proteins on cell death was measured. As can be
seen in FIG. 11, BSO caused cell death of normal lymphocytes as
well as of cells obtained from Friedreich ataxia patients. However,
cells obtained from patients were more sensitive to BSO-induced
oxidative stress, consistent with previous findings. Most
importantly, the various TAT-MTS-FRA fusion proteins, which were
added a few hours before oxidative stress induction, were
demonstrated to partially rescue both normal lymphocytes as well as
patients' cells from cell death. This partial rescue was determined
by both reduction in cell death and by reduction in caspase 3
activity.
[0229] Surprisingly, as also shown in FIG. 11, at least two out of
the three fusion proteins carrying a heterologous MTS (namely,
MTSorf and MTScs) demonstrated a superior protective effect with
respect to the effect demonstrated by the fusion protein carrying
the native MTS.
[0230] In addition, in an independent comparative experiment shown
in FIG. 12, in which the effect of the various TAT-MTS-FRA fusion
proteins on oxidative stress was examined, a fusion protein
carrying another heterologous MTS, namely, MTSlad also demonstrated
a superior protective effect with respect to the effect
demonstrated by the fusion protein carrying the native MTS (FIG.
12C).
[0231] Thus the presently disclosed subject matter further provides
a method for treating or alleviating a mitochondrial disorder, said
method comprising the step of administering to a subject in need of
such treatment a therapeutically effective amount of the fusion
protein as defined herein, thereby treating a mitochondria
disorder.
[0232] In the above and other embodiments, the method for treating
or alleviating a mitochondrial disorder according to the invention
is wherein said functional protein is frataxin or OTC, and the
mitochondrial disorder is Friedreich's ataxia or any other disorder
associated with deficiency of frataxin or defective frataxin or
respectively, a disorder associated with a deficiency of OTC or
with defective OTC.
[0233] The term "treat" or "treatment" or forms thereof as herein
defined means to prevent worsening or arrest or alleviate or cure
the disease or condition in a subject in need thereof. Thus the
term "treatment", "treating" or "alleviating" in the context of the
intention does not refers to complete curing of the diseases, as it
does not change the mutated genetics causing the disease. This term
refers to alleviating at least one of the undesired symptoms
associated with the disease, improving the quality of life of the
subject, decreasing disease-caused mortality, or (if the treatment
in administered early enough) preventing the full manifestation of
the mitochondrial disorder before it occurs, mainly to organs and
tissues that have a high energy demand. The treatment may be a
continuous prolonged treatment for a chronic disease or a single,
several or multiple administrations for the treatment of an acute
condition such as encephalopathy and liver failure that is
accompanied by stormy lactic acidosis, hyperammonemia and
coagulopathy.
[0234] Notably, in the case of metabolic or mitochondrial disorders
there is no need to restore protein activity back to 100%, but
rather raise it above a certain energetic threshold which can vary
from patient to patient depending on basal protein activity.
[0235] In addition, treatment of mitochondrial disorders using
replacement therapy is necessarily more complex than replacement of
a cytosolic gene product and must consider not only the need to
target and cross multiple membranes in mitochondria, but also the
fact that many proteins in the mitochondria act as multi-component
complexes which require appropriate assembly in order to integrate
properly. Additionally, many of the mitochondrial gene defects
cause severe neurologic symptoms as the primary or most prominent
phenotype, and, as mentioned above, drug delivery across the BBB is
difficult.
[0236] Therapeutic formulations may be administered in any
conventional route and dosage formulation. Formulations typically
comprise at least one active ingredient, as defined above, together
with one or more acceptable carriers thereof. Thus, administration
can be any one of intravenous, intraperitoneal, intramuscular and
intratechal administration. Oral administration is also
contemplated.
[0237] The term "therapeutically effective amount" (or amounts) of
the peptide for purposes herein defined is determined by such
considerations as are known in the art in order to cure or at least
arrest or at least alleviate the medical condition.
[0238] As indicated above, in the case of metabolic or
mitochondrial disorders it is not required to restore protein
activity back to 100%. Rather, raising the protein activity to
above a certain energetic threshold, which can vary from patient to
patient depending on basal protein activity, can be sufficient to
provide a therapeutic effect.
[0239] In some embodiments the therapeutically effective amount may
be determined for each patient individually, based on the patient's
basal protein activity. The patient's basal protein activity, or
the level of protein activity may in turn be determined using any
method known in the art, for example, by subjecting a biological
sample obtained from a patient (e.g. blood (white cells), skin
fibroblast cells, whole tissue biopsies) to an in vitro enzymatic
assay or immunohistochemistry or any specific imaging such as
PET.
[0240] In some embodiments the functional human mitochondrial
protein is OTC and the patient's basal OTC activity is determined
by liver biopsy.
[0241] In some embodiments the therapeutically effective amount
according to the presently disclosed subject matter is between
about 0.5 mg/kg to about 2.0 mg/kg body weight of the subject in
need thereof which is the Human Equivalent Dose (HED) of an
effective amount in mice of between about 7 mg/kg to about 25
mg/kg.
[0242] Thus in specific embodiments disclosed is a pharmaceutical
composition as herein defined wherein the therapeutically effective
amount administered is from about 0.5 mg/Kg to about 2 mg/Kg body
weight of said subject.
[0243] As used herein, the term "subject in need" is to be taken to
mean a human suffering from a mitochondrial disorder as herein
defined.
[0244] In the above and other embodiments, the method for treating
or alleviating a mitochondrial disorder according to the invention
further comprises administering an additional therapeutic
agent.
[0245] The term "additional therapeutic agent" as herein defined
refers to any agent that is administered in addition to the fusion
protein according to the invention in order to alleviate the
symptoms associated with the disease or disorder the treatment of
which is desirable. In the above and other embodiments of the
disclosed subject matter, the fusion protein of the invention and
said additional therapeutic agent are administered simultaneously.
Alternatively or additionally, said fusion protein and said
additional therapeutic agent are administered at different time
points, at different intervals between administrations, for
different durations of time, or in a different order.
[0246] The disclosed subject matter further provides a method for
introducing a functional mitochondrial protein into mitochondria of
a subject, said method comprising the step of administering to a
subject in need of such treatment a therapeutically effective
amount of the fusion protein as defined herein, thereby introducing
a functional mitochondrial protein into the mitochondria of a
subject in need thereof.
[0247] In some embodiments the functional human mitochondrial
protein thereby introduced restores at least partial activity of
the wild type human mitochondrial protein, for example at least 5%,
at least 10%, at least 15%, at least 20%, at least 30%, at least
40%, at least 50%, at least 60%, at least 70%, at least 80%, at
least 90% or up to 100% of the activity of a wild type human
mitochondrial protein.
[0248] Thus, the disclosed subject matter further provides a method
for restoring at least in part activity of a defective or deficient
or unfunctional human mitochondrial protein in a subject in need,
by administering a fusion as herein disclosed to said subject. The
human mitochondrial protein may be active per se, or may be a
member of a mitochondrial protein complex.
[0249] By another one of its aspects, the disclosed subject matter
further provides a method for alleviating oxidative stress in a
subject in need thereof, said method comprising the step of
administering to a subject in need of such treatment a
therapeutically effective amount of the fusion protein as herein
defined, thereby alleviating oxidative stress in said subject.
[0250] The term "oxidative stress" as herein defined refers to an
imbalance between the systemic manifestations of reactive oxygen
species (ROS) and an ability of a biological system to readily
detoxify the reactive intermediates or to repair the resulting
damage. Disturbances in the normal redox state of cells can cause
toxic effects through the production of peroxides and free radicals
that damage all components of the cell. In addition, since some
reactive oxidative species act as cellular messengers in redox
signaling, accumulation of ROS can cause disruptions in normal
mechanisms of cellular signaling. In humans, oxidative stress is
thought to be involved in the development of various disorders and
conditions, among which are cancer, Parkinson's disease, Alzheimer
disease to name but a few.
[0251] It is appreciated that certain features of the presently
disclosed subject matter, which are, for clarity, described in the
context of separate embodiments, may also be provided in
combination in a single embodiment. Conversely, various features of
the invention, which are, for brevity, described in the context of
a single embodiment, may also be provided separately or in any
suitable sub combination or as suitable in any other described
embodiment of the invention. Certain features described in the
context of various embodiments are not to be considered essential
features of those embodiments, unless the embodiment is inoperative
without those elements.
[0252] Disclosed and described, it is to be understood that this
invention is not limited to the particular examples, process steps,
and materials disclosed herein as such process steps and materials
may vary somewhat. It is also to be understood that the terminology
used herein is used for the purpose of describing particular
embodiments only and not intended to be limiting since the scope of
the present invention will be limited only by the appended claims
and equivalents thereof.
[0253] It must be noted that, as used in this specification and the
appended claims, the singular forms "a", "an" and "the" include
plural referents unless the content clearly dictates otherwise.
[0254] Throughout this specification and the claims which follow,
unless the context requires otherwise, the word "comprise", and
variations such as "comprises" and "comprising", will be understood
to imply the inclusion of a stated integer or step or group of
integers or steps but not the exclusion of any other integer or
step or group of integers or steps.
[0255] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention is related. The
following terms are defined for purposes of the invention as
described herein.
[0256] The following Examples are representative of techniques
employed by the inventors in carrying out aspects of the present
invention. It should be appreciated that while these techniques are
exemplary of preferred embodiments for the practice of the
invention, those of skill in the art, in light of the present
disclosure, will recognize that numerous modifications can be made
without departing from the spirit and intended scope of the
invention.
EXAMPLES
[0257] Without further elaboration, it is believed that one skilled
in the art can, using the preceding description, utilize the
present invention to its fullest extent. The following preferred
specific embodiments are, therefore, to be construed as merely
illustrative, and not limitative of the claimed invention in any
way.
[0258] Standard molecular biology protocols known in the art not
specifically described herein are generally followed essentially as
in Sambrook & Russell, 2001.
Experimental Procedures
Cell Cultures
[0259] Lymphocytes (Lym 43) and fibroblasts (Fib. 78 and Fib. 65,
F816) from Friedreich's ataxia. patients were obtained from Coriell
Cell Repositories (Camden, N.J.) and grown at the recommended
medium (fibroblasts in Eagle's Minimum Essential Medium (EMEM) with
Earle's salts and non-essential amino acids, and lymphocytes in
Roswell Park Memorial Institute (RPMI) Medium 1640 with 2m M
L-glutamine, all supplemented with 15% fetal bovine serum not
inactivated). Human BJ AB cells (EBV-negative Burkitt's lymphoma
cells; provided by Hanna Ben-Bassat, Hadassah Medical Center,
Jerusalem, Israel) were grown in RPMI medium supplemented with 20%
heat-inactivated fetal calf serum (FCS), 2 mM. L-glutamine, 100
U/mL penicillin, and 100 .mu.g/mL streptomycin. Normal lymphocytes
[20] were grown in RPMI 1640 supplemented with 10% fetal calf serum
(FCS) and antibiotics as above. HepG2 cells (ATCC: HB-8065) were
grown in EMEM supplemented with 10% FCS, 2 mM L-Glutamine,
non-essential amino acids, antibiotics and sodium pyruvate. All
culture cells were grown in 37.degree. C. with 5% CO.sub.2. All
media and supplements were purchased from Biological Industries
(Bet Ha'emek, Israel). The cells were kept in a humidified
atmosphere with 5% CO.sub.2 at 37.degree. C. All cultures were
tested for mycoplasma contamination and were found to be
negative.
Cloning of the Plasmids Encoding the Fusion Proteins
His-TAT-MTSfra-FRA
[0260] The plasmid His-TAT-LAD was prepared as previously reported
[23] and was cut with BamHI and XhoII to remove the LAD sequence,
thus obtaining a vector fragment encoding the His-TAT domain. The
full-length frataxin was generated by PCR using a frataxin clone
(purchased from Open Biosystem. Ltd., clone no. 4842134) as a
template and a pair primers covering the whole sequence, including
its native MTS, 5'-CGCGGATCCGTGGACTCTCGGGCGCCG-3' (forward) and
5'-ACGCTCGAGTCAAGCATCTTTCCGGAATAGGC-3' (reverse), as denoted by SEQ
ID NO. 7 and SEQ ID NO. 8, respectively. The PCR fragment was cut
with BamHI and XhoII and ligated with the vector fragment, thus
obtaining the plasmid encoding the His-TAT-MTSfra-FRA fusion
protein.
His-MTSlad-FRA
[0261] The plasmid encoding the His-TAT-MTSfra-FRA was cut with
BamHI and BsaI to remove the MTSfra sequence. The MTSlad (the
native MTS of lipoamide dehydrogenase) was obtained by PCR using
the plasmid His-TAT-LAD [23] as a template and the following pair
of primers: 5'-CGCGGATCCACAGAGCTGGAGTCGTGTGTA-3' (forward) and
5'-CATAGG-TGGTCTCATCTAGAGAGCCTGGGTGGCCCAAAGTTCCAGATGCGTAAGTTCTCAGAG
GCA-3' (reverse) as denoted by SEQ ID NO. 9 and SEQ ID NO. 10,
respectively. The PCR product was cut with BamHI and BsaI and
ligated to the vector fragment thus obtaining the plasmid encoding
the His-TAT-MTSlad-FRA fusion protein.
His-TAT-MTSorf-FRA
[0262] The plasmid encoding the His-TAT-MTSfra-FRA was cut with
BamHI and BsaI to remove the MTSfra sequence as described above.
The MTSorf (the native MTS of the protein C6ORF66 assembly factor)
was obtained by PCR using the plasmid His-TAT-ORF as a template and
the following pair of primers: 5'-CGCGGATCCGGGAGCACTAGTGATTCGC-3'
(forward) and 5'-CATAGGTG
GTCTCATCTAGAGAGCCTGGGTGGCCCAAAGTTCCAGAAGAGGGGTGTCTGGGAGC GA-3'
(reverse), as denoted by SEQ ID NO. 11 and SEQ ID NO. 12,
respectively. The PCR product was cut with BamHI and BsaI and
ligated to the vector fragment thus obtaining the plasmid encoding
the His-TAT-MTSorf-FRA fusion protein.
His-TAT-MTScs-FRA
[0263] The plasmid encoding the His-TAT-MTSfra-FRA was cut with
BamHI and BsaI to remove the MTSfra sequence as described above.
The MTScs (the native MTS of the protein citrate synthase) was
generated by synthesizing two oligonucleotides covering the MTScs
sequence and the BamHI and BsaI sites at the ends:
5'-GATCCGGCTTACTTACTGCGGCCGCCCGGCTCTTGGGAACCAAGAATGCATCTTGT
CTTGTTCTTGCAGCCCGGCATGCCAGTTCTGGAACTTrGGGCCACCCAGGCTCTC-3'
(forward) and 5'-TCTAGAGAGCCTGGGTGGCCCAAAGTTCCAG
AACTGGCATGCCGGGCTGCAAGAACAAGACAAGATGCATTCTTGGTTCCCAAGAGC
CGGGCGGCCGCAGTAAGTAAAGCCG-3' (reverse), as denoted by SEQ ID NO. 13
and SEQ ID NO. 14, respectively. The oligonucleotides were ligated
to the vector fragment thus obtaining the plasmid encoding the
His-TAT-MTScs-FRA fusion protein. In some of the assays described
below the His-TAT-MTScs-FRA fusion protein construct used was
obtained from Genscript (Lot #342511S03/P10011312; 2.5 mg/ml stock
in 50 mM Tris-HCl, 300 mM NaCl, 10% Glycerol, pH 8.0).
[0264] All plasmids were confirmed by restriction enzymes and
sequencing analyses.
TABLE-US-00001 TABLE 1 Nucleic acid and Amino acid sequences SEQ ID
NO. Sequence Name 1 aggaagaagcggagacagcgacgaaga nt seq. encoding
TAT fragment 2 TGGACTCTCGGGCGCCGCgcagtagccggcctcctggcgtcacccagc nt
seq. of ccggcccaggcccagaccctcacccgggtcccgcggccggcagagttggccccactc
MTSfra tgcggccgccgtggcctgcgcaccgacatcgatgcgacctgcacgccccgccgcgca
agttcgaaccaacgtggcctcaaccagatttggaatgtcaaaaagcagagtgtctat
ttgatgaatttgaggaaa 3 GCTTTACTTACTGCGGCCGCCCGGCTCTTGGGAACCA nt seq.
of AGAATGCATCTTGTCTTGTTCTTGCAGCCCGGCATGCC MTScs AGT 4
ggagcactagtgattcgcggtatcaggaatttcaacctagagaaccgagcggaacgg nt seq.
of gaaatcagcaagatgaagccctctgtcgctcccagacacccctct MTSorf 5
cagagctggagtcgtgtgtactgctccttggccaagagaggccatttcaatcgaata nt seq.
of tctcatggcctacagggactttctgcagtgcctctgagaacttacgca MTSlad 6
tctggaactttgggccacccaggctctctagatgagaccacctatgaaagactagcag nt seq.
of aggaaacgctggactctttagcagagttattgaagaccttgcagacaagccatacacgt
Mature ttgaggactatgatgtctcctttgggagtggtgtcttaactgtcaaactgggtggagatc
Frataxin
taggaacctatgtgatcaacaagcagacgccaaacaagcaaatctggctatcttctccat (FRA)
ccagtggacctaagcgttatgactggactgggaaaaactgggtgtactcccacgacggcg
tgtccctccatgagctgctggccgcagagctcactaaagccttaaaaaccaaactggact
tgtcttCCTTGGCCTATTCCGGAAAAGATGCTTGA 7 CGCGGATCCGTGGACTCTCGGGCGCCG
forward primer for precursor frataxin cloning 8
ACGCTCGAGTCAAGCATCTTTTCCGGAATAGGC reverse primer for precursor
frataxin cloning 9 CGCGGATCCACAGAGCTGGAGTCGTGTGTA forward primer
for MTS lad cloning 10 CATAGGTGGTCTCATCTAGAGAGCCTGGGTGGCCCAA
reverse AGTTCCAGATGCGTAAGTTCTCAGAGGCA primer for MTS lad cloning 11
CGCGGATCCGGGAGCACTAGTGATTCGC forward primer for MTS orf cloning 12
CATAGGTGGTCTCATCTAGAGAGCCTGGGTGGCCCAA reverse
AGTTCCAGAAGAGGGGTGTCTGGGAGCGA primer for MTS orf cloning 13
GATCCGGCTTTACTTACTGCGGCCGCCCGGCTCTTGGG forward
AACCAAGAATGCATCTTGTCTTGTTCTTGCAGCCCGGC oligo for
ATGCCAGTTCTGGAACTTTGGGCCACCCAGGCTCTC MTS cs cloning 14
TCTAGAGAGCCTGGGTGGCCCAAAGTTCCAGAACTGG reverse
CATGCCGGGCTGCAAGAACAAGACAAGATGCATTCTT primer for
GGTTCCCAAGAGCCGGGCGGCCGCAGTAAGTAAAGCC MTS cs G cloning 15
CTGAAGGGCCGTGACCTTCTCACTCTAAGAAACTTTA nt seq. of
CCGGAGAAGAAATTAAATATATGCTATGGCTATCAGC Mature
AGATCTGAAATTTAGGATAAAACAGAAAGGAGAGTA OTC
TTTGCCTTTATTGCAAGGGAAGTCCTTAGGCATGATTT
TTGAGAAAAGAAGTACTCGAACAAGATTGTCTACAGA
AACAGGCTTTGCACTTCTGGGAGGACATCCTTGTTTTC
TTACCACACAAGATATTCATTTGGGTGTGAATGAAAG
TCTCACGGACACGGCCCGTGTATTGTCTAGCATGGCA
GATGCAGTATTGGCTCGAGTGTATAAACAATCAGATT
TGGACACCCTGGCTAAAGAAGCATCCATCCCAATTAT
CAATGGGCTGTCAGATTTGTACCATCCTATCCAGATCC
TGGCTGATTACCTCACGCTCCAGGAACACTATAGCTCT
CTGAAAGGTCTTACCCTCAGCTGGATCGGGGATGGGA
ACAATATCCTGCACTCCATCATGATGAGCGCAGCGAA
ATTCGGAATGCACCTTCAGGCAGCTACTCCAAAGGGT
TATGAGCCGGATGCTAGTGTAACCAAGTTGGCAGAGC
AGTATGCCAAAGAGAATGGTACCAAGCTGTTGCTGAC
AAATGATCCATTGGAAGCAGCGCATGGAGGCAATGTA
TTAATTACAGACACTTGGATAAGCATGGGACAAGAAG
AGGAGAAGAAAAAGCGGCTCCAGGCTTTCCAAGGTTA
CCAGGTTACAATGAAGACTGCTAAAGTTGCTGCCTCT
GACTGGACATTTTTACACTGCTTGCCCAGAAAGCCAG
AAGAAGTGGATGATGAAGTCTTTTATTCTCCTCGATCA
CTAGTGTTCCCAGAGGCAGAAAACAGAAAGTGGACA
ATCATGGCTGTCATGGTGTCCCTGCTGACAGATTACTC ACCTCAGCTCCAGAAGCCTAAATTTTGA
16 ATGTACCGCTACCTGGCCAAAGCGCTGCTGCCGTCCC nt seq. of
GGGCCGGGCCCGCTGCCCTGGGCTCCGCGGCCAACCA MTS of
CTCGGCCGCGTTGCTGGGCCGGGGCCGCGGACAGCCC human
GCCGCCGCCTCGCAGCCGGGGCTCGCATTGGCCGCCC mitochon. GGCGCCACTAC GLUD2
17 MGSSHHHHHHSSGLVPRGSHMRKKRRQRRRGSDPALLT aa seq. of
AAARLLGTKNASCLVLAARHASSGTLGHPGSLDETTYE His TAT
RLAEETLDSLAEFFEDLADKPYTFEDYDVSFGSGVLTVK MTS(cs)
LGGDLGTYVINKQTPNKQIWLSSPSSGPKRYDWTGKNW 81-210
VYSHDGVSLHELLAAELTKALKTKLDLSSLAYSGKDAFRA 18
MGSSHHHHHHSSGLVPRGSHMRKKRRQRRRGSDPWTL aa seq. of
GRRAVAGLLASPSPAQAQTLTRVPRPAELAPLCGRRGLR HTFrataxin
TDIDATCTPRRASSNQRGLNQIWNVKKQSVYLMNLRKS [His
GTLGHPGSLDETTYERLAEETLDSLAEFFEDLADKPYTFE TAT
DYDVSFGSGVLTVKLGGDLGTYVINKQTPNKQIWLSSPS MTS(fra)
SGPKRYDWTGKNWVYSHDGVSLHELLAAELTKALKTK 81-210 LDLSSLAYSGKDA FRA] 19
MGSSHHHHHHSSGLVPRGSHMRKKRRQRRRGSDPQSW aa seq. of
SRVYCSLAKRGHFNRISHGLQGLSAVPLRTYASGTLGHP His TAT
GSLDETTYERLAEETLDSLAEFFEDLADKPYTFEDYDVS MTS(lad)
FGSGVLTVKLGGDLGTYVINKQTPNKQIWLSSPSSGPKR 81-210
YDWTGKNWVYSHDGVSLHELLAAELTKALKTKLDLSS FRA LAYSGKDA 20
MGSSHHHHHHSSGLVPRGSHMRKKRRQRRRGSDPGAL aa seq. of
VIRGRNFNLENRAEREISKMKPSVAPRHPSSGTLGHPGS His TAT
LDETTYERLAEETLDSLAEFFEDLADKPYTFEDYDVSFG MTS(orf)
SGVLTVKLGGDLGTYVINKQTPNKQIWLSSPSSGPKRYD 81-210
WTGKNWVYSHDGVSLHELLAAELTKALKTKLDLSSLA FRA YSGKDA 21 YGRKKRRQRRR aa
seq. of TAT 22 WTLGRRAVAGLLASPSPAQAQTLTRVPRPAELAPLCGR aa seq. of
RGLRTDIDATCTPRRASSNQRGLNQIWNVKKQSVYLMN MTS(fra) LRK 23
ALLTAAARLLGTKNASCLVLAARHAS aa seq. of MTS(cs) 24
QSWSRVYCSLAKRGHFNRISHGLQGLSAVPLRTYA aa seq. of MTS(lad) 25
GALVIRGIRNFNLENRAEREISKMKPSVAPRHPS aa seq. of MTS(orf) 26
SGTLGHPGSLDETTYERLAEETLDSLAEFFEDLADKPYTF aa seq. of
EDYDVSFGSGVLTVKLGGDLGTYVINKQTPNKQIWLSSP Mature
SSGPKRYDWTGKNWVYSHDGVSLHELLAAELTKALKT frataxin KLDLSSLAYSGKDA 27
RKKRRQRRR aa seq. of TAT fragment 28
MRKKRRQRRRGSDPALLTAAARLLGTKNASCLVLAAR aa seq. of
HASSGTLGHPGSLDETTYERLAEETLDSLAEFFEDLADK TAT
PYTFEDYDVSFGSGVLTVKLGGDLGTYVINKQTPNKQI MTS(cs)
WLSSPSSGPKRYDWTGKNWVYSHDGVSLHELLAAELT 81-210 KALKTKLDLSSLAYSGKDA
FRA 29 MRKKRRQRRRGSDPWTLGRRAVAGLLASPSPAQAQTLT aa seq. of
RVPRPAELAPLCGRRGLRTDIDATCTPRRASSNQRGLNQI TAT
WNVKKQSVYLMNLRKSGTLGHPGSLDETTYERLAEETL MTS(fra)
DSLAEFFEDLADKPYTFEDYDVSFGSGVLTVKLGGDLGT 81-210
YVINKQTPNKQIWLSSPSSGPKRYDWTGKNWVYSHDGV FRA
SLHELLAAELTKALKTKLDLSSLAYSGKDA 30
MRKKRRQRRRGSDPQSWSRVYCSLAKRGHFNRISHGLQ aa seq. of
GLSAVPLRTYASGTLGHPGSLDETTYERLAEETLDSLAE TAT
FFEDLADKPYTFEDYDVSFGSGVLTVKLGGDLGTYVINK MTS(lad)
QTPNKQIWLSSPSSGPKRYDWTGKNWVYSHDGVSLHEL 81-210
LAAELTKALKTKLDLSSLAYSGKDA FRA 31
MRKKRRQRRRGSDPGALVIRGIRNFNLENRAEREISKMK aa seq. of
PSVAPRHPSSGTLGHPGSLDETTYERLAEETLDSLAEFFE TAT
DLADKPYTFEDYDVSFGSGVLTVKLGGDLGTYVINKQT MTS(orf)
PNKQIWLSSPSSGPKRYDWTGKNWVYSHDGVSLHELLA 81-210
AELTKALKTKLDLSSLAYSGKDA FRA 32 GSDP aa seq. of linker 33
MRKKRRQRRRALLTAAARLLGTKNASCLVLAARHASS aa seq. of
GTLGHPGSLDETTYERLAEETLDSLAEFFEDLADKPYTFE TAT
DYDVSFGSGVLTVKLGGDLGTYVINKQTPNKQIWLSSPS MTS(cs)
SGPKRYDWTGKNWVYSHDGVSLHELLAAELTKALKTK 81-210 LDLSSLAYSGKDA FRA A
linker 34 MRKKRRQRRRWTLGRRAVAGLLASPSPAQAQTLTRVPR aa seq. of
PAELAPLCGRRGLRTDIDATCTPRRASSNQRGLNQIWNV TAT
KKQSVYLMNLRKSGTLGHPGSLDETTYERLAEETLDSL MTS(fra)
AEFFEDLADKPYTFEDYDVSFGSGVLTVKLGGDLGTYVI 81-210
NKQTPNKQIWLSSPSSGPKRYDWTGKNWVYSHDGVSL FRA
HELLAAELTKALKTKLDLSSLAYSGKDA .DELTA. linker 35
MRKKRRQRRRQSWSRVYCSLAKRGHFNRISHGLQGLSA aa seq. of
VPLRTYASGTLGHPGSLDETTYERLAEETLDSLAEFFEDL TAT
ADKPYTFEDYDVSFGSGVLTVKLGGDLGTYVINKQTPN MTS(lad)
KQIWLSSPSSGPKRYDWTGKNWVYSHDGVSLHELLAAE 81-210 LTKALKTKLDLSSLAYSGKDA
FRA A linker 36 MRKKRRQRRRGALVIRORNFNLENRAEREISKMKPSVA aa seq. of
PRHPSSGTLGHPGSLDETTYERLAEETLDSLAEFFEDLAD TAT
KPYTFEDYDVSFGSGVLTVKLGGDLGTYVINKQTPNKQI MTS(orf)
WLSSPSSGPKRYDWTGKNWVYSHDGVSLHELLAAELT 81-210 KALKTKLDLSSLAYSGKDA
FRA .DELTA. linker
[0265] Table 1 above summarizes the nucleic acid sequences (nt) of
the primers used as described above (i.e. the primers denoted by
SEQ ID NO. 7-SEQ ID NO. 14), the amino acid sequence (aa) of the
TAT domain (denoted by SEQ ID NO. 21), the amino acid sequence of a
fragment of the TAT domain used in the present disclosure (denoted
by SEQ ID NO. 27) and the nucleic acid sequence encoding therefor
(denoted by SEQ ID NO. 1), the amino acid sequences of the various
MTSs, namely MTS fra, MTS cs, MTS orf and MTS lad (denoted by SEQ
ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 25 and SEQ ID NO. 24,
respectively) and the nucleic acid sequences encoding therefor
(denoted by SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO.
5, respectively), the amino acid sequence of mature frataxin
(denoted by SEQ ID NO. 26) and the nucleic acid sequence encoding
therefor (denoted by SEQ ID NO. 6), as well as the nucleic acid
sequences encoding the OTC protein and the MTS of human
mitochondrial GLUD2 (denoted by SEQ ID NO. 15 and SEQ ID NO. 16,
respectively). A four-amino acid long linker, having the amino acid
sequence of GSDP is also listed in Table 1 above and is denoted by
SEQ ID NO. 32.
[0266] In addition Table 1 above indicates the amino acid sequences
of the various His TAT MTS 81-210 FRA constructs, which comprise
the mature frataxin, namely, His TAT MTS(cs) 81-210 FRA, His TAT
MTS(fra) 81-210 FRA (also denoted herein as "HTFrataxin"), His TAT
MTS(lad) 81-210 FRA and His TAT MTS(orf) 81-210 FRA (denoted by SEQ
ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19 and SEQ ID NO. 20,
respectively). The various His TAT MTS 81-210 FRA constructs
denoted by SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19 and SEQ ID
NO. 20 all comprise a short peptide linker denoted by SEQ ID NO.
32, which joins the TAT and MTS domains.
[0267] Table 1 above further indicates the amino acid sequences of
the various fusion constructs constructed without a His tag at
their N-termini and with a short peptide linker denoted by SEQ ID
NO. 32 (which joins the TAT and MTS domains). These constructs
comprise TAT, a linker, MTS and mature frataxin (81-210 FRA),
namely TAT MTS(cs) 81-210 FRA, TAT MTS(fra) 81-210 FRA, TAT
MTS(lad) 81-210 FRA and TAT MTS(orf) 81-210 FRA, denoted by SEQ ID
NO. 28, SEQ ID NO. 29, SEQ ID NO. 30 and SEQ ID NO. 31,
respectively.
[0268] In addition Table 1 above indicates the amino acid sequences
of the various fusion constructs constructed without a His tag and
without a linker. These fusion constructs comprise TAT, MTS and
mature frataxin (81-210 FRA), namely TAT MTS(cs) 81-210 FRA A
linker, TAT MTS(fra) 81-210 FRA A linker, TAT MTS(lad) 81-210 FRA A
linker and TAT MTS(orf) 81-210 FRA A linker, denoted by SEQ ID NO.
33, SEQ ID NO. 34, SEQ ID NO. 35 and SEQ ID NO. 36,
respectively.
Proteins Expression and Purification
[0269] E. coli BL21-CodonPlus (.lamda.DE3) or HMS competent cells
transformed with plasmids encoding the fusion proteins were
incubated at 37.degree. C. in a saline lactose broth (SLB medium)
containing kanamycine (50 .mu.g/ml), tetracycline (12.5 .mu.g/ml)
and chloramphenicol (34 .mu.g/ml). At an OD.sub.600 of 0.2-0.3,
0.1% glycerol and 0.1 mM potassium glutamate were added to the
culture which was then heat-shocked for 20-30 min at 42.degree. C.,
after which the bacteria were grown at 37.degree. C. until an
OD.sub.600 of 0.8. Protein expression was induced by adding
isopropyl-beta-D-thiogalactopyranoside (IPTG, for final
concentrations see Table 2 below). After 18 hrs of incubation at
22.degree. C., the cells were harvested by centrifugation (500 g
for 15 min at 4.degree. C.).
[0270] For the purification procedure, bacteria pellets from 0.5 L
culture of expressed cells were sonicated in binding buffer (PBS,
pH 7.4, 0.4 M NaCl, 10% Glycerol, 1 mM
phenylmethylsulphonylfluoride (PMSF) and 30 mM imidazole
(Sigma-Aldrich, St. Louis, Mo., USA)). The suspensions were
clarified by centrifugation (35,000 g for 30 min at 4.degree. C.),
and the supernatants containing the fusion proteins were purified
under native conditions, using binding buffer pre-equilibrated
HiTrap Chelating HP columns (Amersham-Pharmacia Biotech, Uppsala,
Sweden). Columns were washed by stepwise addition of increasing
imidazole concentrations. Finally, the target proteins were eluted
with elution buffer (PBS, pH 7.4, 0.4 M NaCl, 10% Glycerol, 250 mM
imidazole). All purification procedures were carried out using the
FPLC system AKTA (Amersham-Pharmacia Biotech). Imidazole, NaCl and
glycerol were removed by transferring the purified proteins to PBS
using PD-10 desalting columns (GE Healthcare, Piscataway, N.J.,
USA). Aliquots of the proteins were kept frozen at -80.degree. C.
until use.
TABLE-US-00002 TABLE 2 Expression conditions of TAT-MTS-FRA fusion
proteins Heat Temp, for IPTG Protein Bacterial Host Shock induction
(.degree. C.) (mM) TAT-MTSfra-FRA codonPlus yes 22 over night 0.5
TAT-MTScs-FRA codonPlus yes 22 over night 0.5 TAT-MTSorf-FRA HMS
yes 22 over night 1.0 TAT-MTSlad-FRA HMS yes 22 over night 1.0
Characterization of the Fusion Proteins
Determination of Protein Concentration
[0271] Protein concentration was measured according to the Bradford
method, using the Bradford reagent and the standard curve of BSA.
Protein concentration was determined at a wavelength of 595 nm.
Separation of Proteins by Electrophoresis
[0272] Samples from the various protein fractions (5-20 .mu.g
protein/lane) were loaded on 12% or 15% (w/v) SDS-PAGE gels. The
separation of proteins was done using Sturdier Slab Gel
Electrophoresis apparatus according to the manufacturer's
instructions (Hoefer Sci Instruments, San Francisco, Calif.,
USA).
Western Blot Analysis
[0273] Proteins (5-20 .mu.g protein/lane) were resolved on 12%-15%
SDS-PAGE gels and transferred onto an Immobilon-P Transfer membrane
(Millipore, Bradford, Pa., USA). Western blot analysis was
performed using either anti-frataxin (Abcam), or anti-His
(Amersham-Pharmacia Biotech) antibodies at dilutions of 1:600 and
1:30,000, respectively, to identify the relevant proteins. Primary
antibody binding was detected by blotting with a suitable secondary
antibody conjugated to horseradish peroxidase (HRP) (1:10,000).
Band visualization was done using an enhanced chemiluminescence kit
(EZ-ECL, Biological Industries, Beit-Haemek, Israel).
Internalization of TAT-MTS-FRA into the Mitochondria of Patients'
Fibroblasts
[0274] FA patient fibroblasts (Coriell repository, #GM03816, termed
F816) were thawed and passed at least 2-3 times prior to assay
performance and then incubated in the presence of the fusion
protein construct TAT-MTS(cs)-FRA (20 .mu.g/ml) comprising frataxin
and citrate synthase as the mitochondrial targeting sequence (MTS)
for 2, 6 and 48 hours, with fresh addition of TAT-MTS(CS)-FXN (at
20 .mu.g/ml) after 24 hours. Vehicle was added as negative control.
Cells were fractionated to cytosolic (C) and mitochondrial (M)
fractions using Mitochondria isolation kit (Millipore, MIT1000).
All fractions were analyzed in Western blot analysis using a
monoclonal anti-frataxin antibody directed against the C-terminal
region of frataxin (Abnova, cat. no. H00002395-M02, lot 08078-3F3)
diluted 1:500 in blocking solution. Detection was performed using 1
ml at 1 mg/ml of a goat anti mouse IgG-h+1 alkaline phosphatase
conjugated antibody (BETHYL, Cat#A90-116AP-16) diluted to 1:20,000
in blocking solution. Signal was developed using the ready-to-use
buffered alkaline phosphatase substrate for use in immunoblotting
BCIP /NBT-Blue (Sigma, B3804).
Aconitase Activity Assay
[0275] Aconitase activity assay was performed using a commercial
kit (Abcam, ab109712, microplate assay kit) according to the
manufacturer's instructions. The conversion of isocitrate to
cis-aconitate is measured as an increase in absorbance at OD 240 nm
at specific time points and calculated as activity rate
(mOD/min).
BSO Experiments for Inducing Oxidative Stress
[0276] Normal or patients' lymphocytes (4.times.10.sup.3 cells/100
.mu.l) were seeded in Dulbecco's Modified Eagle Medium (DMEM, Bet
Ha'emek, Israel) medium without phenol red and sodium pyruvate
(experimental medium) and after 3-4 hr the tested TAT-MTS-FRA
fusion protein (at a final concentration of 0.1 .mu.g/.mu.l) was
added to the cells for 5 or 24 hr. Following the incubation time
with the fusion protein, L-Buthionine-sulfoximine (BSO, Sigma
B2640) at different concentrations was added for an additional
period of 48 hr. At the end of the incubation time, cell cultures
were subjected to cell viability or cell proliferation assays,
using the CellTiter-Blue.TM. kit (Promega, Madison, Wis.) according
to manufacturer's instructions, as detailed below. In experiments
conducted in fibroblasts, cells (3.times.10.sup.3 cells/100 .mu.l)
were seeded in the growth medium as specified above and left for 24
hr to allow the cells to adhere. After 24 hr, the medium was
changed to the experimental medium and the experiment was continued
as described for lymphocytes above.
Cell Proliferation Assay
[0277] 10.sup.4/100 .mu.l/well cells growing in suspension or
5.times.10.sup.3/100 .mu.l/well adherent cells were seeded and
treated with increasing concentrations of the fusion protein for
48-72 h, after which cellTiter-Blue.RTM. reagent (Promega, Madison,
Wis., USA) was added according to the manufacturer's instructions
to determine cell survival. All treatments were performed in
triplicates.
In Vitro Caspase 3 Activity Assay
[0278] 10.sup.4/100 .mu.l/well cells growing in suspension or
5.times.10.sup.3/100 .mu.l/well adherent cells were treated with
the fusion protein (0.15 .mu.g/.mu.l, final concentration).
Caspase3 activity within the cells was assessed by Apo-ONE.RTM.
Homogeneous Caspase3/7 Assay Kit (Promega). Caspase 3 activity
assays were carried in parallel to cell viability assays.
Preparation of his-TAT-MTS-OTC Constructs
[0279] Fusion protein constructs comprising ornithine
transcarbamoylase (OTC) conjugated to a His-TAT-MTS fragment,
namely, His-TAT-MTS(otc)-OTC, His-TAT-MTS(lad)-OTC,
His-TAT-MTS(cs)-OTC and His-TAT-MTS(orf)-OTC were obtained from
GenScript. These fusion protein constructs were prepared by
expressing vectors harboring these constructs in E. coli cells and
purified from inclusion bodies to about 80% purity using Ni column
chromatography.
TABLE-US-00003 TABLE 3 Nucleic acid and amino acid sequences of
TAT-MTS-OTC constructs SEQ ID NO. Sequence Name 37
CTGTTTAACCTGCGCATTCTGCTGAACAATGCGGCCTTCCG nt seq. of
TAACGGCCATAATTTTATGGTCCGCAACTTCCGTTGCGGTCA MTS of
GCCGCTGCAAAATAAAGTGCAG OTC 38 LFNLRILLNNAAFRNGHNFMVRNFRCGQPLQNKVQ
aa seq. of MTS of OTC 39 LKGRDLLTLRNFTGEEIKYMLWLSADLKFRIKQKGEYLPLLQ
aa seq. of G KSLGMIFEKRSTRTRLSTETGFALLGGHPCFLTTQDIHLG mature
VNESLTDTARVLSSMADAVLARVYKQSDLDTLAKEASIPIIN OTC
GLSDLYHPIQILADYLTLQEHYSSLKGLTLSWIGDGNNILHS
IMMSAAKFGMHLQAATPKGYEPDASVTKLAEQYAKENGTKLL
LTNDPLEAAHGGNVLITDTWISMGQEEEKKKRLQAFQGYQVT
MKTAKVAASDWTFLHCLPRKPEEVDDEVFYSPRSLVFPEAEN
RKWTIMAVMVSLLTDYSPQLQKPKF 40 CATATGGGCT CATCGCATCA TCATCATCAT nt
seq. of CACTCATCAG GTCTGGTTCC GCGTGGCTCG His-TAT- CACATGTATG
GTCGCAAAAA ACGTCGTCAA MTS(otc)- CGTCGCCGTC TGTTTAACCT GCGCATTCTG
OTC CTGAACAATG CGGCCTTCCG TAACGGCCAT .DELTA. linker AATTTTATGG
TCCGCAACTT CCGTTGCGGT CAGCCGCTGC AAAATAAAGT GCAGCTGAAA GGCCGCGATC
TGCTGACCCT GCGTAACTTC ACGGGTGAAG AAATCAAATA CATGCTGTGG CTGAGCGCAG
ACCTGAAATT CCGCATCAAA CAAAAAGGCG AATACCTGCC GCTGCTGCAG GGCAAATCTC
TGGGTATGAT TTTTGAAAAA CGTAGTACCC GCACGCGTCT GTCCACCGAA ACGGGCTTTG
CCCTGCTGGG CGGTCATCCG TGTTTCCTGA CCACGCAAGA TATCCACCTG GGTGTGAACG
AAAGTCTGAC CGATACGGCA CGCGTTCTGA GCTCTATGGC AGACGCTGTG CTGGCTCGTG
TTTATAAACA GTCCGATCTG GACACCCTGG CGAAAGAAGC CTCAATTCCG ATTATCAATG
GCCTGTCGGA TCTGTACCAT CCGATTCAAA TCCTGGCGGA CTATCTGACC CTGCAGGAAC
ACTACAGTTC CCTGAAAGGT CTGACCCTGA GTTGGATCGG CGATGGTAAC AATATTCTGC
ATAGCATCAT GATGTCTGCA GCTAAATTTG GCATGCACCT GCAAGCGGCC ACCCCGAAAG
GTTATGAACC GGATGCCAGC GTTACGAAAC TGGCAGAACA GTACGCTAAA GAAAACGGTA
CCAAACTGCT GCTGACGAAT GATCCGCTGG AAGCAGCTCA TGGCGGTAAC GTCCTGATTA
CCGACACGTG GATCTCTATG GGCCAGGAAG AAGAAAAGAA AAAACGTCTG CAGGCGTTTC
AAGGTTATCA GGTTACCATG AAAACGGCCA AAGTCGCGGC CAGCGATTGG ACCTTCCTGC
ACTGCCTGCC GCGTAAACCG GAAGAAGTCG ATGACGAAGT GTTTTACTCA CCGCGCTCGC
TGGTGTTCCC GGAAGCAGAA AATCGTAAAT GGACCATCAT GGCTGTTATG GTGTCCCTGC
TGACCGACTA TTCCCCGCAA CTGCAAAAAC CGAAATTCTA ATGAAAGCTT 41
CATATGGGCT CATCTCATCA TCATCATCAT nt seq. of CATTCGTCAG GTCTGGTCCC
GCGTGGCTCT His-TAT- CACATGCGTA AAAAACGTCG TCAGCGTCGT MTS(cs)-
CGTGGCAGTG ATCCGGCACT GCTGACCGCA OTC GCAGCACGTC TGCTGGGTAC
GAAAAACGCT AGCTGCCTGG TGCTGGCTGC GCGTCATGCG TCTGAATTTC TGAAAGGCCG
TGATCTGCTG ACCCTGCGCA ACTTCACGGG TGAAGAAATC AAATACATGC TGTGGCTGAG
TGCCGACCTG AAATTTCGTA TCAAACAAAA AGGCGAATAC CTGCCGCTGC TGCAGGGCAA
ATCCCTGGGT ATGATTTTCG AAAAACGCAG TACCCGTACG CGCCTGTCCA CCGAAACGGG
CTTTGCACTG CTGGGCGGTC ATCCGTGTTT CCTGACCACG CAAGATATCC ACCTGGGTGT
GAACGAATCA CTGACCGATA CGGCTCGTGT TCTGAGCTCT ATGGCAGACG CAGTGCTGGC
ACGTGTTTAT AAACAGTCGG ATCTGGACAC CCTGGCTAAA GAAGCGTCAA TTCCGATTAT
CAATGGCCTG TCGGATCTGT ACCATCCGAT TCAAATCCTG GCGGACTATC TGACCCTGCA
GGAACACTAC AGTTCCCTGA AAGGTCTGAC CCTGAGCTGG ATCGGCGATG GTAACAATAT
TCTGCATAGC ATCATGATGT CTGCCGCAAA ATTTGGCATG CACCTGCAAG CTGCGACCCC
GAAAGGTTAT GAACCGGACG CCAGCGTCAC GAAACTGGCC GAACAGTACG CAAAAGAAAA
CGGTACCAAA CTGCTGCTGA CGAATGATCC GCTGGAAGCC GCACATGGCG GTAACGTTCT
GATTACCGAC ACGTGGATCA GCATGGGCCA GGAAGAAGAA AAGAAAAAAC GTCTGCAGGC
CTTTCAAGGT TATCAGGTTA CCATGAAAAC GGCAAAAGTC GCTGCGTCTG ATTGGACCTT
CCTGCACTGC CTGCCGCGCA AACCGGAAGA AGTCGATGAC GAAGTGTTTT ACTCACCGCG
TTCGCTGGTT TTCCCGGAAG CGGAAAATCG CAAATGGACC ATTATGGCTG TGATGGTCTC
TCTGCTGACG GACTACTCGC CGCAACTGCA AAAACCGAAA TTCTAATGAA AGCTT 42
CATATGGGTT CATCACATCA TCATCATCAT nt seq. of CATTCATCAG GTCTGGTCCC
GCGTGGTTCA His-TAT- CACATGCGTA AAAAACGTCG TCAGCGTCGT MTS(orf)-
CGTGGCAGTG ATCCGGGTGC GCTGGTCATT OTC CGTGGCATCC GCAACTTTAA
TCTGGAAAAC CGTGCGGAAC GCGAAATTAG TAAAATGAAA CCGTCCGTGG CACCGCGTCA
TCCGTCTGAA TTTCTGAAAG GCCGTGATCT GCTGACCCTG CGCAACTTCA CGGGTGAAGA
AATCAAATAC ATGCTGTGGC TGAGTGCAGA CCTGAAATTC CGTATCAAAC AAAAGGGTGA
ATACCTGCCG CTGCTGCAGG GCAAATCCCT GGGTATGATT TTCGAAAAAC GCTCAACCCG
TACGCGCCTG TCGACCGAAA CGGGCTTTGC CCTGCTGGGC GGTCATCCGT GCTTCCTGAC
CACGCAAGAT ATCCACCTGG GTGTGAACGA ATCACTGACC GATACGGCAC GTGTTCTGAG
CTCTATGGCA GACGCAGTGC TGGCTCGTGT TTATAAACAG TCGGATCTGG ACACCCTGGC
AAAAGAAGCT AGCATTCCGA TTATCAATGG CCTGTCTGAT CTGTACCATC CGATTCAAAT
CCTGGCGGAC TATCTGACCC TGCAGGAACA CTACAGTTCC CTGAAAGGTC TGACCCTGAG
CTGGATCGGC GATGGTAACA ATATTCTGCA TAGCATCATG ATGTCTGCGG CCAAATTCGG
CATGCACCTG CAAGCAGCTA CCCCGAAAGG TTATGAACCG GACGCCTCCG TTACGAAACT
GGCGGAACAG TACGCCAAAG AAAACGGCAC CAAACTGCTG CTGACGAATG ATCCGCTGGA
AGCGGCCCAT GGCGGTAACG TCCTGATTAC CGACACGTGG ATCAGCATGG GCCAGGAAGA
AGAAAAGAAA AAACGTCTGC AGGCATTTCA AGGTTATCAG GTTACCATGA AAACGGCTAA
AGTCGCAGCT TCTGATTGGA CCTTCCTGCA CTGTCTGCCG CGCAAACCGG AAGAAGTCGA
TGACGAAGTG TTTTACTCAC CGCGTTCGCT GGTGTTCCCG GAAGCGGAAA ATCGCAAATG
GACCATTATG GCTGTGATGG TGTCGCTGCT GACGGACTAC TCGCCGCAAC TGCAAAAACC
GAAATTCTAA TGAAAGCTT 43 CATATGGGTA GTTCACATCA TCATCATCAT nt seq. of
CACTCGTCGG GTCTGGTGCC GCGTGGCTCA His-TAT- CACATGCGTA AAAAACGTCG
TCAGCGTCGT MTS(lad)- CGTGGCTCAG ATCCGCAATC ATGGTCGCGC OTC
GTCTATTGCT CGCTGGCGAA ACGTGGTCAT TTTAACCGCA TTAGCCACGG CCTGCAGGGT
CTGTCTGCAG TGCCGCTGCG TACCTACGCT GAATTTCTGA AAGGCCGTGA TCTGCTGACC
CTGCGCAACT TCACGGGTGA AGAAATCAAA TACATGCTGT GGCTGAGCGC AGACCTGAAA
TTTCGTATCA AACAAAAAGG CGAATACCTG CCGCTGCTGC AGGGCAAATC TCTGGGTATG
ATTTTCGAAA AACGCTCAAC CCGTACGCGC CTGTCGACCG AAACGGGCTT TGCCCTGCTG
GGCGGTCATC CGTGTTTCCT GACCACGCAG GATATCCACC TGGGTGTGAA CGAAAGTCTG
ACCGATACGG CACGTGTTCT GAGCTCTATG GCAGACGCAG TGCTGGCTCG TGTTTATAAA
CAGTCCGATC TGGACACCCT GGCAAAAGAA GCTAGTATTC CGATTATCAA TGGCCTGTCC
GATCTGTACC ATCCGATTCA AATCCTGGCG GACTATCTGA CCCTGCAGGA ACACTACAGT
TCCCTGAAAG GTCTGACGCT GAGCTGGATC GGCGATGGTA ACAATATTCT GCATAGTATC
ATGATGTCCG CGGCCAAATT CGGCATGCAC CTGCAAGCAG CTACCCCGAA AGGTTATGAA
CCGGACGCCT CTGTTACGAA ACTGGCGGAA CAGTACGCCA AAGAAAACGG TACCAAACTG
CTGCTGACGA ATGATCCGCT GGAAGCGGCC CATGGCGGTA ACGTCCTGAT TACCGACACG
TGGATCAGTA TGGGCCAGGA AGAAGAAAAG AAAAAACGTC TGCAGGCGTT TCAAGGTTAT
CAGGTTACCA TGAAAACGGC CAAAGTCGCA GCTAGCGATT GGACCTTCCT GCACTGCCTG
CCGCGCAAAC CGGAAGAAGT CGATGACGAA GTGTTTTATA GCCCGCGTTC TCTGGTGTTC
CCGGAAGCGG AAAATCGCAA ATGGACCATC ATGGCCGTTA TGGTGTCGCT GCTGACCGAT
TACTCCCCGC AACTGCAAAA ACCGAAATTC TAATGAAAGC TT 44 MGSSHHHHHH
SSGLVPRGSH MYGRKKRRQR aa seq. of RRLFNLRILL NNAAFRNGHN FMVRNFRCGQ
His-TAT- PLQNKVQLKG RDLLTLRNFT GEEIKYMLWL MTS(otc)- SADLKFRIKQ
KGEYLPLLQG KSLGMIFEKR OTC STRTRLSTET GFALLGGHPC FLTTQDIHLG .DELTA.
linker VNESLTDTAR VLSSMADAVL ARVYKQSDLD TLAKEASIPI INGLSDLYHP
IQILADYLTL QEHYSSLKGL TLSWIGDGNN ILHSIMMSAA KFGMHLQAAT PKGYEPDASV
TKLAEQYAKE NGTKLLLTND PLEAAHGGNV LITDTWISMG QEEEKKKRLQ AFQGYQVTMK
TAKVAASDWT FLHCLPRKPE EVDDEVFYSP RSLVFPEAEN RKWTIMAVMV SLLTDYSPQL
QKPKF 45 MGSSHHHHHH SSGLVPRGSH MRKKRRQRRR aa seq. of GSDPALLTAA
ARLLGTKNAS CLVLAARHAS His-TAT- EFLKGRDLLT LRNFTGEEIK YMLWLSADLK
MTS(cs)- FRIKQKGEYL PLLQGKSLGM IFEKRSTRTR OTC LSTETGFALL GGHPCFLTTQ
DIHLGVNESL TDTARVLSSM ADAVLARVYK QSDLDTLAKE ASIPIINGLS DLYHPIQILA
DYLTLQEHYS SLKGLTLSWI GDGNNILHSI MMSAAKFGMH LQAATPKGYE PDASVTKLAE
QYAKENGTKL LLTNDPLEAA HGGNVLITDT WISMGQEEEK KKRLQAFQGY QVTMKTAKVA
ASDWTFLHCL PRKPEEVDDE VFYSPRSLVF PEAENRKWTI MAVMVSLLTD YSPQLQKPKF
46 MGSSHHHHHH SSGLVPRGSH MRKKRRQRRR aa seq. of GSDPGALVIR
GIRNFNLENR AEREISKMKP His-TAT- SVAPRHPSEF LKGRDLLTLR NFTGEEIKYM
MTS(orf)- LWLSADLKFR IKQKGEYLPL LQGKSLGMIF OTC EKRSTRTRLS
TETGFALLGG HPCFLTTQDI HLGVNESLTD TARVLSSMAD AVLARVYKQS DLDTLAKEAS
IPIINGLSDL YHPIQILADY LTLQEHYSSL KGLTLSWIGD GNNILHSIMM SAAKFGMHLQ
AATPKGYEPD ASVTKLAEQY AKENGTKLLL TNDPLEAAHG GNVLITDTWI SMGQEEEKKK
RLQAFQGYQV TMKTAKVAAS DWTFLHCLPR KPEEVDDEVF YSPRSLVFPE AENRKWTIMA
VMVSLLTDYS PQLQKPKF 47 MGSSHHHHHH SSGLVPRGSH MRKKRRQRRR aa seq. of
GSDPQSWSRV YCSLAKRGHF NRISHGLQGL His-TAT- SAVPLRTYAE FLKGRDLLTL
RNFTGEEIKY MTS(lad)- MLWLSADLKF RIKQKGEYLP LLQGKSLGMI OTC
FEKRSTRTRL STETGFALLG GHPCFLTTQD IHLGVNESLT DTARVLSSMA DAVLARVYKQ
SDLDTLAKEA SIPIINGLSD LYHPIQILAD YLTLQEHYSS LKGLTLSWIG DGNNILHSIM
MSAAKFGMHL QAATPKGYEP DASVTKLAEQ YAKENGTKLL LTNDPLEAAH GGNVLITDTW
ISMGQEEEKK KRLQAFQGYQ VTMKTAKVAA SDWTFLHCLP RKPEEVDDEV FYSPRSLVFP
EAENRKWTIM AVMVSLLTDY SPQLQKPKF 48 RKKRRQRRRL FNLRILLNNA AFRNGHNFMV
aa seq. of RNFRCGQPLQ NKVQLKGRDL LTLRNFTGEE TAT- IKYMLWLSAD
LKFRIKQKGE YLPLLQGKSL MTS(otc)- GMIFEKRSTR TRLSTETGFA LLGGHPCFLT
OTC TQDIHLGVNE SLTDTARVLS SMADAVLARV .DELTA. linker YKQSDLDTLA
KEASIPIING LSDLYHPIQI LADYLTLQEH YSSLKGLTLS WIGDGNNILH SIMMSAAKFG
MHLQAATPKG YEPDASVTKL AEQYAKENGT KLLLTNDPLE AAHGGNVLIT DTWISMGQEE
EKKKRLQAFQ GYQVTMKTAK
VAASDWTFLH CLPRKPEEVD DEVFYSPRSL VFPEAENRKW TIMAVMVSLL TDYSPQLQKPKF
49 MRKKRRQRRR GSDPALLTAA ARLLGTKNAS aa seq. of CLVLAARHAS
EFLKGRDLLT LRNFTGEEIK TAT- YMLWLSADLK FRIKQKGEYL PLLQGKSLGM
MTS(cs)- IFEKRSTRTR LSTETGFALL GGHPCFLTTQ OTC DIHLGVNESL TDTARVLSSM
ADAVLARVYK QSDLDTLAKE ASIPIINGLS DLYHPIQILA DYLTLQEHYS SLKGLTLSWI
GDGNNILHSI MMSAAKFGMH LQAATPKGYE PDASVTKLAE QYAKENGTKL LLTNDPLEAA
HGGNVLITDT WISMGQEEEK KKRLQAFQGY QVTMKTAKVA ASDWTFLHCL PRKPEEVDDE
VFYSPRSLVF PEAENRKWTI MAVMVSLLTD YSPQLQKPKF 50 MRKKRRQRRR
GSDPGALVIR GIRNFNLENR aa seq. of AEREISKMKP TAT- SVAPRHPSEF
LKGRDLLTLR NFTGEEIKYM MTS(orf)- LWLSADLKFR IKQKGEYLPL LQGKSLGMIF
OTC EKRSTRTRLS TETGFALLGG HPCFLTTQDI HLGVNESLTD TARVLSSMAD
AVLARVYKQS DLDTLAKEAS IPIINGLSDL YHPIQILADY LTLQEHYSSL KGLTLSWIGD
GNNILHSIMM SAAKFGMHLQ AATPKGYEPD ASVTKLAEQY AKENGTKLLL TNDPLEAAHG
GNVLITDTWI SMGQEEEKKK RLQAFQGYQV TMKTAKVAAS DWTFLHCLPR KPEEVDDEVF
YSPRSLVFPE AENRKWTIMA VMVSLLTDYS PQLQKPKF 51 MRKKRRQRRR GSDPQSWSRV
YCSLAKRGHF aa seq. of NRISHGLQGL TAT- SAVPLRTYAE FLKGRDLLTL
RNFTGEEIKY MTS(lad)- MLWLSADLKF RIKQKGEYLP LLQGKSLGMI OTC
FEKRSTRTRL STETGFALLG GHPCFLTTQD IHLGVNESLT DTARVLSSMA DAVLARVYKQ
SDLDTLAKEA SIPIINGLSD LYHPIQILAD YLTLQEHYSS LKGLTLSWIG DGNNILHSIM
MSAAKFGMHL QAATPKGYEP DASVTKLAEQ YAKENGTKLL LTNDPLEAAH GGNVLITDTW
ISMGQEEEKK KRLQAFQGYQ VTMKTAKVAA SDWTFLHCLP RKPEEVDDEV FYSPRSLVFP
EAENRKWTIM AVMVSLLTDY SPQLQKPKF 52 MRKKRRQRRR ALLTAA ARLLGTKNAS
CLVLAARHAS aa seq. of EFLKGRDLLT LRNFTGEEIK YMLWLSADLK TAT-
FRIKQKGEYL PLLQGKSLGM IFEKRSTRTR MTS(cs)- LSTETGFALL GGHPCFLTTQ
DIHLGVNESL OTC TDTARVLSSM ADAVLARVYK QSDLDTLAKE linker ASIPIINGLS
DLYHPIQILA DYLTLQEHYS SLKGLTLSWI GDGNNILHSI MMSAAKFGMH LQAATPKGYE
PDASVTKLAE QYAKENGTKL LLTNDPLEAA HGGNVLITDT WISMGQEEEK KKRLQAFQGY
QVTMKTAKVA ASDWTFLHCL PRKPEEVDDE VFYSPRSLVF PEAENRKWTI MAVMVSLLTD
YSPQLQKPKF 53 MRKKRRQRRR GALVIR GIRNFNLENR AEREISKMKP aa seq. of
SVAPRHPSEF LKGRDLLTLR NFTGEEIKYM TAT- LWLSADLKFR IKQKGEYLPL
LQGKSLGMIF MTS(orf)- EKRSTRTRLS TETGFALLGG HPCFLTTQDI OTC .DELTA.
HLGVNESLTD TARVLSSMAD AVLARVYKQS linker DLDTLAKEAS IPIINGLSDL
YHPIQILADY LTLQEHYSSL KGLTLSWIGD GNNILHSIMM SAAKFGMHLQ AATPKGYEPD
ASVTKLAEQY AKENGTKLLL TNDPLEAAHG GNVLITDTWI SMGQEEEKKK RLQAFQGYQV
TMKTAKVAAS DWTFLHCLPR KPEEVDDEVF YSPRSLVFPE AENRKWTIMA VMVSLLTDYS
PQLQKPKF 54 MRKKRRQRRR QSWSRV YCSLAKRGHF NRISHGLQGL aa seq. of
SAVPLRTYAE FLKGRDLLTL RNFTGEEIKY TAT- MLWLSADLKF RIKQKGEYLP
LLQGKSLGMI MTS(lad)- FEKRSTRTRL STETGFALLG GHPCFLTTQD OTC .DELTA.
IHLGVNESLT DTARVLSSMA DAVLARVYKQ linker SDLDTLAKEA SIPIINGLSD
LYHPIQILAD YLTLQEHYSS LKGLTLSWIG DGNNILHSIM MSAAKFGMHL QAATPKGYEP
DASVTKLAEQ YAKENGTKLL LTNDPLEAAH GGNVLITDTW ISMGQEEEKK KRLQAFQGYQ
VTMKTAKVAA SDWTFLHCLP RKPEEVDDEV FYSPRSLVFP EAENRKWTIM AVMVSLLTDY
SPQLQKPKF
[0280] Table 3 recites the amino acid sequence of the MTS of the
OTC protein and the nucleotide sequence encoding therefor (denoted
by SEQ ID NO. 38 and by SEQ ID NO. 37, respectively), the amino
acid sequence of mature OTC (denoted by SEQ ID NO. 39), and the
nucleotide sequences encoding the fusion protein constructs
His-TAT-MTS(otc)-OTC .DELTA. linker, His-TAT-MTS(cs)-OTC,
His-TAT-MTS(orf)-OTC and His-TAT-MTS(lad)-OTC (denoted by SEQ ID
NO. 40, denoted by SEQ ID NO. 41, SEQ ID NO. 42 and by SEQ ID NO.
43, respectively). Table 3 also indicates the amino acid sequences
of the fusion protein constructs His-TAT-MTS(otc)-OTC .DELTA.
linker, His-TAT-MTS(cs)-OTC, His-TAT-MTS(orf)-OTC and
His-TAT-MTS(lad)-OTC (denoted by SEQ ID NO. 44, SEQ ID NO. 45, SEQ
ID NO. 46 and by SEQ ID NO. 47, respectively).
[0281] Also recited are fusion protein constructs comprising the
mitochondrial active protein OTC where the fusion protein construct
does not include a His tag, namely the constructs TAT-MTS(otc)-OTC
.DELTA. linker having the amino acid sequences denoted by SEQ ID
NO. 48, TAT-MTS(cs)-OTC (denoted by SEQ ID NO. 49),
TAT-MTS(orf)-OTC (denoted by SEQ ID NO. 50) and TAT-MTS(lad)-OTC
(denoted by SEQ ID NO. 51).
[0282] The fusion protein constructs according to the present
disclosure may be prepared in the presence of a linker situated
between the TAT and the MTS fragments or in its absence. Thus table
3 also recites the sequences of the fusion protein constructs
termed His-TAT-MTS(otc)-OTC .DELTA. linker, TAT-MTS(cs)-OTC .DELTA.
linker, TAT-MTS(orf)-OTC .DELTA. linker, TAT-MTS(lad)-OTC .DELTA.
linker (having the amino acid sequences denoted by SEQ ID NO. 44,
SEQ ID NO. 52, SEQ ID NO. 53 and SEQ ID NO. 54, respectively).
Internalization of his-TAT-MTS-OTC Constructs into the
Mitochondria
[0283] A culture of 10.sup.7 HepG2 cells (ATCC, #HB-8065) was
seeded in T-75 flasks. The next day, TAT-MTS-OTC fusion protein
constructs comprising either OTC, CS or LAD at 12 .mu.g/ml prepared
as described above were added to the cells in Eagle's Minimum
Essential Medium (EMEM) complete medium, or in EMEM medium
supplemented with DMSO (1%) and Trehalose (30 mM) or in EMEM medium
supplemented with DMSO (1%), Trehalose (30 mM) and Ornithine (1 mM)
for an incubation of 20 min, 1 or 3 hours. Cells were then
fractionated to cytosolic and mitochondrial fractions using
Mitochondria isolation kit (Millipore, MIT1000) according to the
manufacturer's instructions. Fractions were analyzed in Western
blot analysis using a monoclonal antibody directed to the C
terminal region of OTC (Aviva Systems Biology, cat. no.
ARP41767_P050), at 1 mg/ml diluted 1:1000. Detection was performed
with the goat anti rabbit IgG (H_L) secondary antibody peroxidase
conjugated (Jackson AffiniPure, Code 111-035-003) at 0.8 mg/ml,
diluted 1:20,000 in blocking buffer ( ). Signal was developed with
chemiluminescence mixture (SC-2048 Santa Cruz) according to the
manufacturer's instructions.
In Vitro Production of Citrulline by the Enzymatic Activity of
OTC
[0284] Purified TAT-MTS-OTC fusion protein constructs comprising
the MTS of OTC or CS prepared as described above were tested for
their enzymatic activity as described in [40]. Briefly, proteins
were diluted in a mitochondria lysis buffer (0.5% Triton, 10 mM
HEPES, pH 7.2 and 2 mM dithiothreitol) to a concentration of 20
.mu.g in a volume of 80 .mu.l. Then the volume of each of the
reaction mixtures was completed to 600 .mu.l by adding 520 .mu.l
reaction mixture (5 mM ornithine, 15 mM carbamoyl phosphate, and
270 mM triethanolamine, pH 7.7). Following vortexing, each reaction
mixture was divided into two separate tubes, the first tube was
incubated at 37.degree. C. for 30 min and the second tube served as
"no-reaction" background and used to subtract endogenous signal of
OTC proteins. Reactions were terminated by adding 150 .mu.l of 1:3
sulfuric acid/phosphoric acid (by volume). Citrulline production
was then determined by adding 20 .mu.l of 3% 2,3-butanedione
monoxime, incubating at 100.degree. C. in the dark for 15 min, and
measuring absorbance at 490 nm. Commercially available OTC from
Bacillus subtilis (Sigma, 5 ng) was used as Positive Control (data
not shown).
Enzymatic Activity of OTC in HepG2 Cells in the Presence of
Ammonia
[0285] HepG2 cells 2.5.times.10.sup.5 (ATCC, #HB-8065) per well
were seeded in two 6-well plates. The next day, TAT-MTS-OTC fusion
protein constructs comprising the MTS of OTC or CS (prepared as
described above) were applied to the cells for 72 hours with
replenishment after each 24 hours. The final concentration of the
fusion proteins was 14 .mu.g/ml, in 3 ml complete medium and the
final buffer dilution was 1:85. Proteins storage buffer
(1.times.PBS, 0.5% Sodium Lauroyl Sarcosine at pH 7.4) served as
vehicle control and untreated cells as negative control. At 48
hours prior to the termination of the assay, cells were treated
with 0.5% serum growth medium in the absence or in the presence of
5 mM Ammonium chloride. Cells viability was monitored for 4 hours
with alamarBlue Cell Viability Assay Reagent (Pierce). Fluorescence
signal was obtained by excitation at 544 nm and emission at 590
nm.
Example 1
Cloning of Plasmids Encoding TA T-MTS-FRA Fusion Proteins
[0286] Expression plasmids encoding the TAT-fusion proteins were
cloned and prepared by standard molecular biology tools known in
the art. For a general reference see Molecular Cloning: A
Laboratory manual (2001) Joseph Sambrook and David William Russell.
TAT [9] having the amino acid sequence as denoted by SEQ ID NO. 27
(encoded by the nucleic acid sequence denoted by SEQ ID NO. 1) was
fused (N-terminal) to the mature human frataxin protein (having the
amino acid sequence as denoted by SEQ ID NO. 26 and encoded by the
nucleic acid sequence as denoted by SEQ ID NO. 6).
[0287] Various fusion constructs were prepared, which differ in
their mitochondrial targeting sequence present at the N terminus of
human frataxin (and thus located between the TAT and mature human
frataxin described above), being either the native mitochondrial
targeting sequence of frataxin (referred to herein as MTSfra,
having the amino acid sequence as denoted by SEQ ID NO. 22 and
encoded by the nucleic acid sequence as denoted by SEQ ID NO. 2) or
other defined MTSs of human mitochondrial proteins, including
lipoamide dehydrogenase (referred to herein as MTSlad, having the
amino acid sequence as denoted by SEQ ID NO. 24 and encoded by the
nucleic acid sequence as denoted by SEQ ID NO. 5), C6ORF66
(referred to herein as MTSorf, having the amino acid sequence as
denoted by SEQ ID NO. 25 and encoded by the nucleic acid sequence
as denoted by SEQ ID NO. 4) and citrate synthase (referred to
herein as MTScs, having the amino acid sequence as denoted by SEQ
ID NO. 23 and encoded by the nucleic acid sequence as denoted by
SEQ ID NO. 3). The various TAT-MTS-FRA fusion proteins are
summarized in Table 4 below and schematically presented in FIG.
1.
[0288] All plasmids were cloned with His-tag at the 5'-terminus of
the coding sequence and all coding sequences were under the control
of the T7 promotor. All clones were confirmed by restriction
enzymes and sequencing analyses.
TABLE-US-00004 TABLE 4 The cloned plasmid constructs No. Plasmid
name Abbreviated name 1 His-TAT-MTSfra-FRA FRA 2 His-TAT-MTSlad-FRA
(lad)FRA 3 His-TAT-MTSorf-FRA (orf)FRA 4 His-TAT-MTScs-FRA (cs)FRA
Abbreviations: lad, Lipoamide dehydrogenase protein (E3 subunit);
orf, C6ORF66 assembly factor; and cs, citrate synthase.
[0289] As indicated above, the amino acid sequences of the fusion
proteins obtained from the cloned plasmid constructs indicated as
1-4 in Table 4 above are denoted by SEQ ID NO. 18, SEQ ID NO. 19,
SEQ ID NO. 20 and SEQ ID NO. 17, respectively.
Example 2
[0290] Expression of the Fusion Proteins in E. coli Hosts
[0291] Expression of the fusion proteins was performed in E. coli
hosts as described below and was calibrated for optimal expression
conditions. As known in the art, there are several different
bacterial expression systems. Successful expression of recombinant
proteins is often dependent on the strain of the bacteria
expression system used. Thus, for each fusion protein prepared as
described above, four different E. coli bacterial stains were
tested: BL21-CodonPlus, BL21, Rosetta and HMS (Invitrogen, USA) and
the host for expression was thereby selected. The conditions for
expression were also calibrated for each of the TAT-fusion
proteins, by changing several parameters, including the
concentration of the inducer Isopropyl
.beta.-D-1-thiogalactopyranoside (IPTG) and length of induction
growth conditions (i.e. temperature, addition of chemicals,
etc.)
[0292] Upon expression, bacterial cells were disrupted and cellular
sub-fractions were prepared, separating the soluble and non-soluble
fractions. Analysis was performed for the whole-cell bacteria (W.C.
or whole-cell extract), the soluble fraction (Sol) and insoluble
fraction (Insol) on sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) gels in order to examine whether the
fusion protein was expressed and at which sub-cellular fraction it
accumulates. The goal was to obtain high expression levels of the
different TAT-fusion proteins in the soluble sub-fraction of the
expressing bacteria, for future purification. The different
TAT-fusion proteins were also characterized by Western blots
analyses using both anti-His and anti-frataxin antibodies. Table 2
above summarizes the bacterial host, IPTG concentration and
temperature for production of each TAT-MTS-FRA fusion proteins
carrying a different MTS sequence.
[0293] Typical expression and sub-cellular localization of each of
the four fusion proteins as characterized by SDS-PAGE gels and
Western blots using anti-His antibodies are demonstrated in FIG. 2
and in FIG. 3. Expression of the His-TAT-MTSfra-FRA fusion protein
is shown in FIG. 2A-2B, Expression of the His-TAT-MTSorf-FRA fusion
protein is shown in FIG. 2C-2D, Expression of the
His-TAT-MTSlad-FRA fusion protein is shown in FIG. 3A-3B and
Expression of the His-TAT-MTScs-FRA fusion protein is shown in FIG.
3C-3D. These experiments confirmed the full-length expression of
the different TAT-fusion proteins and their identity.
[0294] It should be pointed out the anti-His antibodies recognize,
most probably, the various fusion proteins with different efficacy,
depending on the exposure or availability of the His sequence in
the final protein preparation, for antibody interactions. Thus,
expression levels of the various fusion proteins are determined
based on the SDS-PAGE gels.
[0295] As can be seen in FIG. 2 and in FIG. 3, the TAT-MTSfra-FRA
fusion protein (FIG. 2A & FIG. 2B) was expressed at low levels
in the bacterial hosts, as compared to the other three fusion
proteins carrying an heterologous MTS (FIG. 2C & FIG. 2D and
FIG. 3A-3D), even under the best calibrated conditions. Thus, using
a heterologous MTS instead of the native frataxin-MTS has an
advantage in its expression levels in a bacterial host. This has
major implications on its future development for human use, where
large quantities of the fusion protein are needed to be produced
(see also below).
Example 3
Purification of the TA T-MTS-FRA Fusion Proteins
[0296] The soluble fractions of the expressed TAT-MTS-FRA fusion
proteins were loaded onto a nickel-chelating column to
affinity-purify these proteins, as detailed above. Calibration
experiments were performed for each of the fusion proteins,
including specific conditions for binding of the fusion protein
onto the affinity column, its elution and removal of the imidazole
from the final protein preparations. One typical purification run
of each of the fusion proteins is demonstrated in FIG. 4 (for
His-TAT-MTSfra-FRA and His-TAT-MTSorf-FRA) and in FIG. 5 (for
His-TAT-MTSlad-FRA and His-TAT-MTScs-FRA).
[0297] For example, FIG. 4A shows an image of an affinity
chromatography purification profile obtained for the fusion protein
TAT-MTSfra-FRA. As can be seen in FIG. 4B, which is an image of
SDS-PAGE analysis of the purification steps of this protein
construct, the protein fraction shown in FIG. 4B, lane 7,
represents the fusion protein TAT-MTSfra-FRA that was eluted from
the column at 250 mM imidazole.
[0298] Corresponding analyses are shown for TAT-MTSorf-FRA in FIG.
4C and FIG. 4D. for TAT-MTSlad-FRA in FIG. 5A and FIG. 5B and for
TAT-MTScs-FRA corresponding analyses are shown in FIG. 5C and FIG.
5D.
[0299] As shown in FIG. 6A, eluted proteins showed a major band of
the expected size (approximately 20-27 kDa) and were >95% pure,
as determined by SDS-PAGE analysis and by Western blot analyses
using both anti-His (FIG. 6B) and anti-Fra antibodies (FIG. 6C). As
can be seen in FIG. 6, TAT-MTS-FRA fusion proteins carrying a
heterologous MTS were full-length, intact proteins with no evidence
for protein degradation (see lane 2 for TAT-MTScs-FRA, lane 3 for
TAT-TSlad-FRA and lane 4 for TAT-MTSorf-FRA). However,
TAT-MTSfra-FRA carrying the native MTS sequence was partially
degraded (see lane 1 in FIG. 6B and in FIG. 6C). Thus, using a
heterologous MTS sequence for the TAT-FRA fusion protein has an
advantage also for keeping the fusion protein intact and
stable.
[0300] In addition, when comparing the total amounts and
concentrations of each of the fusion proteins, which were produced
from the same starting volume of bacterial cultures, fusion
proteins carrying an heterologous MTS were produced in larger
amounts and at higher concentration as compared to that of fusion
protein carrying the native MTS (Table 5, below). This has again
major implications on its future development for human use, where
large quantities of the fusion protein are needed and at high
concentrations. Moreover, the stability of the produced fusion
protein has an additional advantage.
TABLE-US-00005 TABLE 5 TAT-MTS-FRA fusion proteins Final
concentration Amount purified from 0.5 L Protein (mg/ml) bacterial
culture (mg) TAT-MTSfra-FRA 0.2 0.6-0.7 TAT-MTScs-FRA 1.0 3-4
TAT-MTSorf-FRA 1.0 3-4 TAT-MTSlad-FRA 0.8 2.4-3.2
Example 4
Internalization of TA T-MTS-FRA Fusion Proteins
[0301] In order to test the ability of the fusion protein to reach
the mitochondria within intact cells, human BJAB cells were
incubated with one of the purified fusion proteins, namely,
TAT-MTSlad-FRA. After incubation, sub-cellular fractions were
prepared. to separate the mitochondria and the cytosol. The
mitochondria were then treated with proteinase K (Sigma P2308) to
digest proteins nonspecifically adsorbed to the outer membrane,
thereby ensuring that the mitochondrial extract represented only
proteins within the mitochondria. Samples were then analyzed by
Western blot assay for the presence of the FRA-based fusion
protein, using both anti-His and anti-FRA antibodies (see FIG.
7).
[0302] As shown in FIG. 7A, using anti-His antibodies the results
indicated the presence of the His-tagged TAT-MTSlad-FRA fusion
protein within the mitochondrial fractions of the treated cells
after 1 and 5 hours of incubation (lane 4 and lanes 6 & 7,
respectively).
[0303] As shown in FIG. 7B, using anti-FRA antibodies, the presence
of a frataxin fusion protein construct inside the mitochondria is
indicated. From lane 2 of FIG. 7B it is evident that control cells
that were not treated with the fusion protein have endogenous FRA
protein, most probably, both the mature isoform and the
intermediate isoform (indicated by the two arrows).
[0304] As shown in FIG. 7B (lanes 4. 6, 7), upon treatment with the
TAT-MTSlad-FRA fusion protein, the relative amounts of both the
unprocessed fusion protein as well as of the mature protein
increased with the incubation time (FIG. 7B; upper and lower bands,
respectively). Without wishing to be bound by theory, the increase
in the upper band is explained by fusion protein construct that
entered the mitochondria and was not processed.
[0305] Internalization of fusion proteins comprising frataxin into
the mitochondria of cells was examined also for other TAT-MTS-FRA
fusion proteins, namely, TAT-MTSfra-FRA, TAT-MTScs-FRA, and
TAT-MTSorf-FRA (as well as for TAT-MTSlad-FRA), as shown in FIG.
8A. In this experiment, cells were incubated for 3 hours with each
of the TAT-MTS-FRA fusion proteins (at a final concentration of
0.02 .mu.g/.mu.l). The cells were then washed, their mitochondria
were isolated and then submitted to Western blot analysis using
monoclonal anti-FRA antibodies (Abnova, Taiwan; 1 .mu.g/ml) that
are directed to the C-terminus of the frataxin protein.
[0306] As demonstrated in FIG. 8A, lanes 2-5, a slight size
variation was observed among the various fusion protein constructs,
resulting from the variation in the length of the various MTS
polypeptides (see FIG. 1).
[0307] As shown in FIG. 8A, internalization into mitochondria was
observed for all four TAT-MTS-FRA fusion proteins. Remarkably, as
shown in lane 3 of FIG. 8A, among the fusion proteins with a MTS
that is heterologous to frataxin, the citrate synthase MTS (MTScs)
was demonstrated to be delivered most efficiently into the
mitochondria.
[0308] As indicated above, there is a slight variation in the
lengths of the fusion protein constructs that entered the
mitochondria, indicating that under the present experimental
conditions (namely incubation of cells for 3 hours with each of the
TAT-MTS-FRA fusion proteins) the frataxin protein was not cleaved
from its respective His-TAT-MTS-frataxin construct inside the
mitochondria (protein constructs that still include intact
His-TAT-MTS-frataxin are termed below as "un-processed fusion
construct").
[0309] Notably, FIG. 8A also shows that while for the fusion
proteins comprising an MTS which is heterologous to frataxin,
namely the MTS of citrate synthase (cs), lipoamide dehydrogenase
(lad) and C6ORF66 (orf), only a single band (representing the
un-processed fusion construct) was demonstrated inside the
mitochondria, two distinct bands appeared for the fusion protein
comprising the native MTS of frataxin (FIG. 8A, lane 2). Without
wishing to be bound by theory, this may be the result of
instability of the intact fusion protein comprising the TAT and
native frataxin MTS regions.
[0310] The antibodies used to detect frataxin in the
internalization assay presented in FIG. 7B were polyclonal
antibodies directed against human frataxin. These antibodies
recognize the full-length, unprocessed human frataxin very well, as
well as its processed products. In FIG. 8 referred to below,
detection was performed using monoclonal antibodies specifically
directed against the C-terminus of the frataxin protein. As these
monoclonal antibodies are less efficient in recognizing the
protein, particularly at the amounts of protein detected in FIG. 8,
the results of FIG. 7 and FIG. 8 cannot be compared.
[0311] Control Western blot analysis using anti-E1.alpha.
antibodies (Molecular Probes, Eugene, Oreg.) at a dilution of
1:1,000) are demonstrated in FIG. 8B.
Example 5
His-TA T-MTS(Cs)-FRA Enter the Mitochondria of Patients'
Flbroblasts
[0312] Further to the above results demonstrating the ability of
the frataxin fusion proteins to enter mitochondria of human BJAB
cells, the internalization of a frataxin fusion protein comprising
the MTS of citrate synthase into the mitochondria of fibroblasts
obtained from Friedreich's ataxia patients was examined, as
detailed below.
[0313] Fibroblasts obtained from Friedreich's ataxia patients were
treated as described above and incubated in the presence of
His-TAT-MTS(cs)-FRA (at 20 .mu.g/ml) for 2, 6 and 48 hours, with
fresh addition after 24 hours. Mitochondrial and cytosolic
fractions of the fibroblasts were analyzed by Western blot analysis
as described above.
[0314] As demonstrated in FIG. 9, the fusion protein construct
His-TAT-MTS(cs)-FRA was detected in mitochondrial fractions of
fibroblasts after an incubation of 2, 6 and 48 hours. The two bands
detected by the anti-frataxin antibody (indicated by the upper and
lower arrows in FIG. 9) indicate that the fusion protein was
processed inside mitochondria. Frataxin was not detected in the
cytosolic fractions of the fibroblasts indicating that all of the
His-TAT-MTS(cs)-FRA fusion protein construct has entered
mitochondria.
Example 6
[0315] Aconitase Activity in Fibroblasts Obtained from Patients
Following Administration of his-TAT-MTS(Cs)-FRA
[0316] It has been reported that the reduction in the levels of
frataxin within the mitochondria has two direct effects on several
tissue types, namely impaired formation of iron-sulfur (Fe--S)
clusters and a rise in intracellular reactive oxygen species (ROS)
[35]. The decrease in Fe--S containing proteins, such as heme,
electron transport chain (ETC) complexes I-III and the Kreb's cycle
protein aconitase severely impairs cellular respiration [36], which
is further complicated by simultaneous oxidative damage to these
mitochondrial proteins. These events all culminate in an inability
of the mitochondria to fulfill the cell's energy requirements
resulting in cell death [35].
[0317] In order to assess the activity of frataxin inside
mitochondria, the level of aconitase activity in mitochondria
obtained from Friedreich's ataxia patients was examined upon
administration of His-TAT-MTS(cs)-FRA.
[0318] Aconitase activity (mOD/min) was measured by following the
conversion of isocitrate to cis-aconitate, as described above, in
mitochondrial fractions of fibroblasts (F816) obtained from
Friedreich's ataxia patients that were treated as described above
and incubated in the presence of His-TAT-MTS(cs)-FRA or vehicle for
48 hours (FXN or VEH, respectively). His-TAT-MTS(cs)-FRA was
administered at 20 .mu.g/ml and an additional dose of 20 .mu.g/ml
was administered 24 hours after the first administration). HepG2
whole cells homogenate served as positive control (POS.CON).
[0319] As shown in FIG. 10, aconitase activity was higher in
fibroblasts that were incubated with His-TAT-MTS(cs)-FRA (FXN,
14.5) compared to fibroblasts that were incubated in the presence
of the vehicle control (VEH, 10.5). This finding is a clear
demonstration that the fusion protein not only enters mitochondria,
but is also active and available for its various cellular
activities.
[0320] HepG2 whole cells homogenate served as positive control
(POS.CON).
Example 7
[0321] TAT-MTS-FRA Fusion Proteins Partially Rescue FA-Patients'
Cells as Well as Normal Cells from Oxidative Stress
[0322] As indicated above, the reduction in the levels of frataxin
within the mitochondria leads to a rise in intracellular reactive
oxygen species (ROS) [35].
[0323] L-Buthionine sulphoximine (BSO) is an inhibitor of
gamma-glutamylcysteine synthetase (gamma-GCS) and, consequently
lowers tissue glutathione (GSH) concentrations. GSH plays an
important role in cellular defense or protection against a wide
variety of toxic electrophiles via the formation of thioether
conjugates. Therefore, BSO assay was used to inhibit de novo
glutathione synthesis, depleting an important component of these
cells' intrinsic defenses against reactive oxygen species (ROS) and
allowing for the accumulation of ROS produced by natural cell
processes, known to result in cell death [37]. The mechanism by
which BSO inhibits production of GSH and results in cell death was
described by Richardson, T. E. et al. [37]. Because they are
lacking in Frataxin, Friedreich ataxia cells are extremely
sensitive to BSO-induced oxidative stress compared with normal
cells [37], and thus are used as an in vitro model of the long-term
consequences of absent Frataxin.
[0324] Oxidative stress was induced with various concentrations of
BSO in patients' cells as well as in normal healthy cells and the
effect of the various TAT-MTS-FRA fusion proteins on cell death was
measured. As can be seen in FIG. 11, BSO caused cell death of
normal lymphocytes as well as of cells obtained from Friedreich
ataxia patients. However, patients' cells were more sensitive to
BSO-induced oxidative stress, consistent with previous findings,
showing higher percentages of cell death. Most importantly, the
various TAT-MTS-FRA fusion proteins, which were added a few hours
before oxidative stress induction, were demonstrated to partially
rescue both normal lymphocytes as well as patients' cells from cell
death. This partial rescue was determined by both reduction in cell
death and by reduction in caspase 3 activity, as demonstrated in
FIG. 11B.
[0325] As shown in FIG. 11A and FIG. 11B, at least two out of the
three fusion proteins carrying a heterologous MTS (namely, MTSorf
and MTScs) demonstrated a superior protective effect with respect
to the effect demonstrated by the fusion protein carrying the
native MTS, in both patients' cells as well as in healthy cells,
from BSO-induced oxidative stress.
[0326] A comparative study of the ability of the various
TAT-MTS-FRA fusion proteins to rescue BSO-induced oxidative stress
of patients' fibroblasts performed as detailed above is shown in
FIG. 12A-FIG. 12D for the fusion protein constructs TAT-MTSfra-FRA,
TAT-MTScs-FRA, TAT-MTSlad-FRA and TAT-MTSorf-FRA, respectively.
[0327] Interestingly, as demonstrated in FIG. 12B and in FIG. 12C,
respectively, two of the protein constructs comprising heterologous
MTS, namely the TAT-MTScs-FRA and TAT-MTSlad fusion proteins were
more efficient than the fusion protein comprising the native
frataxin MTS (TAT-MTSfra-FRA) in partially rescuing BSO-induced
oxidative stress of patients' fibroblasts.
[0328] Similar protective effects in patients' cells induced with
various concentrations of BSO were also observed for TAT-MTSorf-FRA
and TAT-MTSlad fusion proteins when assayed alone (FIG. 13 and FIG.
14, respectively).
Example 8
Pharmacodynamic and Pharmacokinetic Assessment of TAT-MTS-FRA
Fusion Proteins in Friedreich's Ataxia Mice
[0329] In an ongoing study, in order to evaluate pharmacodynamic,
pharmacokinetic (PK/PD) and safety of TAT-MTS-FRA fusion proteins
in a Friedreich's ataxia preclinical mouse model, the mouse model
JR#18299 FVB; B6-Tg(FXN)1Sars Fxn.sub.tm1MKn/J is used in an
ongoing study. The information obtained from these studies will be
used for determining dose and time interval for further clinical
studies. Briefly, the study requires 45 female mice, 6-8 weeks of
age that are homozygous for the targeted mutation at the mouse
Frataxin locus and hemizygous for the transgene. It is noted that
strain 18299 is the original Sarsero mouse on a mixed genetic
background, which was selected over the original C57BL/6J congenic
strain due to its superior breeding performance and since no
behavioral phenotyping is performed in this study.
[0330] In order to perform the preclinical study, two doses of
TAT-MTScs-FRA fusion protein (at 100 .mu.g and 400 .mu.g) are
administered twice per week for a maximal period of three weeks
along with a vehicle, via tail vein injection into 18229
FVB;B6-Tg(FXN)1Sars Fxntm1Mkn/J mice. Mice are sacrificed 1, 4, 7,
14 or 21 days post injection and then blood is collected by cardiac
puncture and various tissues are harvested (e.g. brain, heart,
liver and kidney). Brain and heart tissue are processed to measure
aconitase activity. Brain and heart tissue are also processed to
measure frataxin levels by ELISA assay in mitochondrial extracts.
Blood, liver and kidney are flash-frozen and stored.
[0331] The effect of the treatment is then assessed in the
harvested tissues.
Example 9
[0332] TAT-MTS-OTC Protein Constructs Internalize into
Mitochondria
[0333] Fusion protein constructs comprising OTC fused to a
His-TAT-MTS were prepared as described above. As shown in FIG. 16,
purified fusion protein constructs comprising the native MTS of
OTC, the MTS of citrate synthase, the MTS of C6ORF66 and the MTS of
lipoamide dehydrogenase were obtained (FIG. 16A, FIG. 16B, FIG. 16C
and FIG. 16D, respectively). The size of the purified fusion
protein constructs obtained was about 40 kDa as determined based on
comparing their gel migration to the migration of the protein
marker shown in FIG. 16E). Similar expression yields were obtained
for all four fusion protein constructs that were prepared.
[0334] In order to examine the ability of the various OTC fusion
protein constructs to enter mitochondria, HepG2 cells were
incubated in the presence of the OTC fusion proteins constructs
comprising either the native MTS of OTC or an MTS that is
heterologous to OTC, namely, His-TAT-MTSotc-OTC, His-TAT-MTScs-OTC
and TAT-MTSlad-OTC as described above.
[0335] First, as shown in FIG. 17A, it is noteworthy that the
fusion protein constructs His-TAT-MTSotc-OTC, His-TAT-MTScs-OTC and
His-TAT-MTSlad-OTC (and His-TAT-MTSorf-OTC, data not shown) were
soluble under the various assay conditions based on the fact that
no precipitation or degradation products were detected.
[0336] Analysis of mitochondrial fractions of HepG2 cells incubated
with the above constructs revealed that the OTC fusion protein
construct comprising the native MTS of OTC (His-TAT-MTSotc-OTC) as
well as the OTC fusion protein constructs comprising an MTS that is
heterologous to the OTC protein, namely citrate synthase (cs) and
lipoamide dehydrogenase (lad) were able to enter mitochondria after
an incubation period of 1 hour (FIG. 17A). FIG. 17A also shows that
prolonging the incubation period to 3 hours resulted in an increase
in the level of the fusion protein construct inside the
mitochondria.
[0337] Interestingly, by comparing the ability of the protein
construct comprising the native MTS of OTC (His-TAT-MTSotc-OTC) to
the ability of OTC fusion protein constructs comprising an MTS that
is heterologous to the OTC protein (namely His-TAT-MTScs-OTC and
His-TAT-MTSlad-OTC) for example upon incubation of 3 hours in EMEM,
it appears that the internalization ability of the fusion protein
constructs comprising an MTS that is heterologous to OTC was
slightly higher than the internalization ability of the fusion
protein construct comprising the native MTS of OTC.
[0338] In order to further confirm the internalization of the OTC
fusion protein constructs into mitochondria, the cytosolic
fractions of the cells incubated with the various His-TAT-MTS-OTC
protein constructs were also analyzed. As shown in FIG. 18A and
FIG. 18B, no fusion protein constructs were observed in any of the
assayed cytosolic fractions.
Example 10
In Vitro Enzymatic Activity of OTC
[0339] As indicated above, OTC is a protein having enzymatic
activity that catalyzes the reaction between carbamoyl phosphate
and ornithine to form citrulline and phosphate. In order to assess
the activity of the purified fusion protein constructs comprising
OTC described herein above, the production of citrulline from
ornithine and carbamoyl phosphate by various fusion protein
constructs was evaluated, as described above.
[0340] As shown in FIG. 19, the fusion protein construct
His-TAT-MTScs-OTC assayed had relatively low enzymatic activity,
where the fusion protein construct His-TAT-MTSotc-OTC had none.
Without wishing to be bound by theory, this may be due to the fact
that OTC is probably not present in the reaction mixture at its
native trimeric state, which is necessary for its enzymatic
activity, since it is conjugated to a His-TAT-MTS fragment.
[0341] However, as shown in FIG. 19 a low level of enzymatic
activity was indeed observed for the fusion protein construct
comprising the MTS of CS.
Example 11
[0342] Enzymatic Activity of OTC in HepG2 Cells Suffering from
Ammonia Stress
[0343] OTC deficiency is the most common urea cycle disorder in
humans. OTC, the defective enzyme in this disorder, is the final
enzyme in the proximal portion of the urea cycle, and is
responsible for converting carbamoyl phosphate and ornithine into
citrulline, as indicated above. In severely affected individuals,
ammonia concentrations increase rapidly causing ataxia, lethargy
and death without rapid intervention.
[0344] In order to test the ability of the OTC fusion protein
constructs to rescue ammonia stress in cells, HepG2 cells were
administered daily (for 3 days) with a fusion protein construct
comprising the native MTS of OTC or with fusion protein construct
comprising the MTS of CS then treated with ammonium chloride, and
finally, their cell viability was evaluated using the alamarBlue
indicator, as described above.
[0345] As shown in FIG. 20, the level of cell viability in the
presence of fusion protein constructs comprising OTC and citrate
synthase as the MTS was similar to the level of cell viability for
cells which were not exposed to ammonium chloride (untreated
cells). Furthermore, the level of cell viability in the presence of
a fusion protein construct comprising OTC and citrate synthase as
the MTS was higher from the level of cell viability in cells
treated with the fusion protein construct comprising the native MTS
of OTC.
[0346] Taken together, the above results demonstrate that fusion
protein constructs comprising TAT, MTS and OTC and in particular
such fusion protein which comprise an MTS which is heterologous to
OTC are able to enter mitochondria, the OTC is then processed,
thereby enabling its enzymatic activity.
[0347] The results also indicated that the fusion protein
constructs without ammonia were not cytotoxic (data not shown).
Example 12
Kinetics of Liver Entry and Dose Determination of TAT-MTS-OTC
Fusion Protein Constructs in Mice
[0348] In order to assess the kinetics of liver entry of OTC, liver
levels of any of the TAT-MTS-OTC fusion protein constructs
described herein are determined in OTC-deficient spf.sup.ash mice
(3 hemizygous males and 3 homozygous females at group) 4, 8, 24 and
48 hours after administration of a fusion protein construct
comprising OTC (500 .mu.g/mouse). Liver pieces and other tissues
(e.g. skeletal muscle, brain) are then harvested and analyzed for
their OTC protein levels by Western blot analysis. In addition, the
activity of OTC is examined in harvested liver, by using an OTC
enzymatic assay (for example the citrulline assay described
above).
[0349] Blood is collected by cardiac puncture and plasma is
isolated for liver function tests (ALT/AST), amino acids (including
citrulline), ammonia determination and OTC protein levels (Western
blot). Urine is collected for orotic acid analysis.
[0350] Male mice are used for further studies in case the above
experiments do not indicate any differences between males and
females.
[0351] Then, protein levels are determined in tissues of mice
administered with various doses of a TAT-MTS-OTC fusion protein
construct (100-500 .mu.g/mouse) at the time point for which the
highest OTC protein levels were detected in the above assay.
Example 13
Phenotype Protection by TAT-MTS-OTC Fusion Protein Constructs
[0352] OTC-deficient spf.sup.ash mice (6 males or 3 males and 3
females) are administered with OTC-shRNA knockdown rAAV according
to Cunningham, S. C. et al. [41]. At a designated time-point (based
on the activity of the OTC-shRNA batch and results obtained from
the above experiments) dosing with a TAT-MTS-OTC fusion protein
construct is commenced by administering 0, 1, 2 or 3 dose(s) per
week to each tested group of mice.
[0353] Analysis is performed by first measuring baseline plasma
levels of ammonia, amino acids (including citrulline) and ALT/AST
as well as urinary orotic acid prior to injection of OTC-shRNA
knockdown rAAV (blood is collected via tail vein nicking). Blood
and urine are collected again prior to administration of a
TAT-MTS-OTC fusion protein construct for the above analyses and
then weekly for the duration of the experiment.
[0354] Mice are monitored and sacrificed upon observation of
clinical signs of hyperammonaemia (lethargy, tremors, ataxia) or 1
month post-injection of a TAT-MTS-OTC fusion protein construct. At
the termination of the experiment, liver, muscle and brain are
frozen in liquid nitrogen or fixed in 4% PFA for vector copy
analysis (quantitative PCR), OTC activity (in liver lysate and
frozen sections) and protein levels (Western blot).
Sequence CWU 1
1
54127DNAHuman immunodeficiency virus type 1 1aggaagaagc ggagacagcg
acgaaga 272237DNAHomo sapiens 2tggactctcg ggcgccgcgc agtagccggc
ctcctggcgt cacccagccc ggcccaggcc 60cagaccctca cccgggtccc gcggccggca
gagttggccc cactctgcgg ccgccgtggc 120ctgcgcaccg acatcgatgc
gacctgcacg ccccgccgcg caagttcgaa ccaacgtggc 180ctcaaccaga
tttggaatgt caaaaagcag agtgtctatt tgatgaattt gaggaaa 237378DNAHomo
sapiens 3gctttactta ctgcggccgc ccggctcttg ggaaccaaga atgcatcttg
tcttgttctt 60gcagcccggc atgccagt 784102DNAHomo sapiens 4ggagcactag
tgattcgcgg tatcaggaat ttcaacctag agaaccgagc ggaacgggaa 60atcagcaaga
tgaagccctc tgtcgctccc agacacccct ct 1025105DNAHomo sapiens
5cagagctgga gtcgtgtgta ctgctccttg gccaagagag gccatttcaa tcgaatatct
60catggcctac agggactttc tgcagtgcct ctgagaactt acgca 1056393DNAHomo
sapiens 6tctggaactt tgggccaccc aggctctcta gatgagacca cctatgaaag
actagcagag 60gaaacgctgg actctttagc agagtttttt gaagaccttg cagacaagcc
atacacgttt 120gaggactatg atgtctcctt tgggagtggt gtcttaactg
tcaaactggg tggagatcta 180ggaacctatg tgatcaacaa gcagacgcca
aacaagcaaa tctggctatc ttctccatcc 240agtggaccta agcgttatga
ctggactggg aaaaactggg tgtactccca cgacggcgtg 300tccctccatg
agctgctggc cgcagagctc actaaagcct taaaaaccaa actggacttg
360tcttccttgg cctattccgg aaaagatgct tga 393727DNAArtificial
Sequenceforward primer for precursor frataxin cloning 7cgcggatccg
tggactctcg ggcgccg 27833DNAArtificial Sequencereverse primer for
precursor frataxin cloning 8acgctcgagt caagcatctt ttccggaata ggc
33930DNAArtificial Sequenceforward primer for MTS lad cloning
9cgcggatcca cagagctgga gtcgtgtgta 301066DNAArtificial
Sequencereverse primer for MTS lad cloning 10cataggtggt ctcatctaga
gagcctgggt ggcccaaagt tccagatgcg taagttctca 60gaggca
661128DNAArtificial Sequenceforward primer for MTS orf cloning
11cgcggatccg ggagcactag tgattcgc 281266DNAArtificial
Sequencereverse primer for MTS orf cloning 12cataggtggt ctcatctaga
gagcctgggt ggcccaaagt tccagaagag gggtgtctgg 60gagcga
6613112DNAArtificial Sequenceforward oligo for MTS cs cloning
13gatccggctt tacttactgc ggccgcccgg ctcttgggaa ccaagaatgc atcttgtctt
60gttcttgcag cccggcatgc cagttctgga actttgggcc acccaggctc tc
11214112DNAArtificial Sequencereverse primer for MTS cs cloning
14tctagagagc ctgggtggcc caaagttcca gaactggcat gccgggctgc aagaacaaga
60caagatgcat tcttggttcc caagagccgg gcggccgcag taagtaaagc cg
11215957DNAHomo sapiens 15ctgaagggcc gtgaccttct cactctaaga
aactttaccg gagaagaaat taaatatatg 60ctatggctat cagcagatct gaaatttagg
ataaaacaga aaggagagta tttgccttta 120ttgcaaggga agtccttagg
catgattttt gagaaaagaa gtactcgaac aagattgtct 180acagaaacag
gctttgcact tctgggagga catccttgtt ttcttaccac acaagatatt
240catttgggtg tgaatgaaag tctcacggac acggcccgtg tattgtctag
catggcagat 300gcagtattgg ctcgagtgta taaacaatca gatttggaca
ccctggctaa agaagcatcc 360atcccaatta tcaatgggct gtcagatttg
taccatccta tccagatcct ggctgattac 420ctcacgctcc aggaacacta
tagctctctg aaaggtctta ccctcagctg gatcggggat 480gggaacaata
tcctgcactc catcatgatg agcgcagcga aattcggaat gcaccttcag
540gcagctactc caaagggtta tgagccggat gctagtgtaa ccaagttggc
agagcagtat 600gccaaagaga atggtaccaa gctgttgctg acaaatgatc
cattggaagc agcgcatgga 660ggcaatgtat taattacaga cacttggata
agcatgggac aagaagagga gaagaaaaag 720cggctccagg ctttccaagg
ttaccaggtt acaatgaaga ctgctaaagt tgctgcctct 780gactggacat
ttttacactg cttgcccaga aagccagaag aagtggatga tgaagtcttt
840tattctcctc gatcactagt gttcccagag gcagaaaaca gaaagtggac
aatcatggct 900gtcatggtgt ccctgctgac agattactca cctcagctcc
agaagcctaa attttga 95716159DNAHomo sapiens 16atgtaccgct acctggccaa
agcgctgctg ccgtcccggg ccgggcccgc tgccctgggc 60tccgcggcca accactcggc
cgcgttgctg ggccggggcc gcggacagcc cgccgccgcc 120tcgcagccgg
ggctcgcatt ggccgcccgg cgccactac 15917190PRTArtificial SequenceHis
TAT MTS(cs) 81-210 FRA 17Met Gly Ser Ser His His His His His His
Ser Ser Gly Leu Val Pro1 5 10 15Arg Gly Ser His Met Arg Lys Lys Arg
Arg Gln Arg Arg Arg Gly Ser 20 25 30Asp Pro Ala Leu Leu Thr Ala Ala
Ala Arg Leu Leu Gly Thr Lys Asn 35 40 45Ala Ser Cys Leu Val Leu Ala
Ala Arg His Ala Ser Ser Gly Thr Leu 50 55 60Gly His Pro Gly Ser Leu
Asp Glu Thr Thr Tyr Glu Arg Leu Ala Glu65 70 75 80Glu Thr Leu Asp
Ser Leu Ala Glu Phe Phe Glu Asp Leu Ala Asp Lys 85 90 95Pro Tyr Thr
Phe Glu Asp Tyr Asp Val Ser Phe Gly Ser Gly Val Leu 100 105 110Thr
Val Lys Leu Gly Gly Asp Leu Gly Thr Tyr Val Ile Asn Lys Gln 115 120
125Thr Pro Asn Lys Gln Ile Trp Leu Ser Ser Pro Ser Ser Gly Pro Lys
130 135 140Arg Tyr Asp Trp Thr Gly Lys Asn Trp Val Tyr Ser His Asp
Gly Val145 150 155 160Ser Leu His Glu Leu Leu Ala Ala Glu Leu Thr
Lys Ala Leu Lys Thr 165 170 175Lys Leu Asp Leu Ser Ser Leu Ala Tyr
Ser Gly Lys Asp Ala 180 185 19018243PRTArtificial
SequenceHTFrataxin [His TAT MTS(fra)81-210 FRA] 18Met Gly Ser Ser
His His His His His His Ser Ser Gly Leu Val Pro1 5 10 15Arg Gly Ser
His Met Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Ser 20 25 30Asp Pro
Trp Thr Leu Gly Arg Arg Ala Val Ala Gly Leu Leu Ala Ser 35 40 45Pro
Ser Pro Ala Gln Ala Gln Thr Leu Thr Arg Val Pro Arg Pro Ala 50 55
60Glu Leu Ala Pro Leu Cys Gly Arg Arg Gly Leu Arg Thr Asp Ile Asp65
70 75 80Ala Thr Cys Thr Pro Arg Arg Ala Ser Ser Asn Gln Arg Gly Leu
Asn 85 90 95Gln Ile Trp Asn Val Lys Lys Gln Ser Val Tyr Leu Met Asn
Leu Arg 100 105 110Lys Ser Gly Thr Leu Gly His Pro Gly Ser Leu Asp
Glu Thr Thr Tyr 115 120 125Glu Arg Leu Ala Glu Glu Thr Leu Asp Ser
Leu Ala Glu Phe Phe Glu 130 135 140Asp Leu Ala Asp Lys Pro Tyr Thr
Phe Glu Asp Tyr Asp Val Ser Phe145 150 155 160Gly Ser Gly Val Leu
Thr Val Lys Leu Gly Gly Asp Leu Gly Thr Tyr 165 170 175Val Ile Asn
Lys Gln Thr Pro Asn Lys Gln Ile Trp Leu Ser Ser Pro 180 185 190Ser
Ser Gly Pro Lys Arg Tyr Asp Trp Thr Gly Lys Asn Trp Val Tyr 195 200
205Ser His Asp Gly Val Ser Leu His Glu Leu Leu Ala Ala Glu Leu Thr
210 215 220Lys Ala Leu Lys Thr Lys Leu Asp Leu Ser Ser Leu Ala Tyr
Ser Gly225 230 235 240Lys Asp Ala19199PRTArtificial SequenceHis TAT
MTS(lad) 81-210 FRA 19Met Gly Ser Ser His His His His His His Ser
Ser Gly Leu Val Pro1 5 10 15Arg Gly Ser His Met Arg Lys Lys Arg Arg
Gln Arg Arg Arg Gly Ser 20 25 30Asp Pro Gln Ser Trp Ser Arg Val Tyr
Cys Ser Leu Ala Lys Arg Gly 35 40 45His Phe Asn Arg Ile Ser His Gly
Leu Gln Gly Leu Ser Ala Val Pro 50 55 60Leu Arg Thr Tyr Ala Ser Gly
Thr Leu Gly His Pro Gly Ser Leu Asp65 70 75 80Glu Thr Thr Tyr Glu
Arg Leu Ala Glu Glu Thr Leu Asp Ser Leu Ala 85 90 95Glu Phe Phe Glu
Asp Leu Ala Asp Lys Pro Tyr Thr Phe Glu Asp Tyr 100 105 110Asp Val
Ser Phe Gly Ser Gly Val Leu Thr Val Lys Leu Gly Gly Asp 115 120
125Leu Gly Thr Tyr Val Ile Asn Lys Gln Thr Pro Asn Lys Gln Ile Trp
130 135 140Leu Ser Ser Pro Ser Ser Gly Pro Lys Arg Tyr Asp Trp Thr
Gly Lys145 150 155 160Asn Trp Val Tyr Ser His Asp Gly Val Ser Leu
His Glu Leu Leu Ala 165 170 175Ala Glu Leu Thr Lys Ala Leu Lys Thr
Lys Leu Asp Leu Ser Ser Leu 180 185 190Ala Tyr Ser Gly Lys Asp Ala
19520198PRTArtificial SequenceHis TAT MTS(orf) 81-210 FRA 20Met Gly
Ser Ser His His His His His His Ser Ser Gly Leu Val Pro1 5 10 15Arg
Gly Ser His Met Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Ser 20 25
30Asp Pro Gly Ala Leu Val Ile Arg Gly Ile Arg Asn Phe Asn Leu Glu
35 40 45Asn Arg Ala Glu Arg Glu Ile Ser Lys Met Lys Pro Ser Val Ala
Pro 50 55 60Arg His Pro Ser Ser Gly Thr Leu Gly His Pro Gly Ser Leu
Asp Glu65 70 75 80Thr Thr Tyr Glu Arg Leu Ala Glu Glu Thr Leu Asp
Ser Leu Ala Glu 85 90 95Phe Phe Glu Asp Leu Ala Asp Lys Pro Tyr Thr
Phe Glu Asp Tyr Asp 100 105 110Val Ser Phe Gly Ser Gly Val Leu Thr
Val Lys Leu Gly Gly Asp Leu 115 120 125Gly Thr Tyr Val Ile Asn Lys
Gln Thr Pro Asn Lys Gln Ile Trp Leu 130 135 140Ser Ser Pro Ser Ser
Gly Pro Lys Arg Tyr Asp Trp Thr Gly Lys Asn145 150 155 160Trp Val
Tyr Ser His Asp Gly Val Ser Leu His Glu Leu Leu Ala Ala 165 170
175Glu Leu Thr Lys Ala Leu Lys Thr Lys Leu Asp Leu Ser Ser Leu Ala
180 185 190Tyr Ser Gly Lys Asp Ala 1952111PRTHuman immunodeficiency
virus type 1 21Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg1 5
102279PRTHomo sapiens 22Trp Thr Leu Gly Arg Arg Ala Val Ala Gly Leu
Leu Ala Ser Pro Ser1 5 10 15Pro Ala Gln Ala Gln Thr Leu Thr Arg Val
Pro Arg Pro Ala Glu Leu 20 25 30Ala Pro Leu Cys Gly Arg Arg Gly Leu
Arg Thr Asp Ile Asp Ala Thr 35 40 45Cys Thr Pro Arg Arg Ala Ser Ser
Asn Gln Arg Gly Leu Asn Gln Ile 50 55 60Trp Asn Val Lys Lys Gln Ser
Val Tyr Leu Met Asn Leu Arg Lys65 70 752326PRTHomo sapiens 23Ala
Leu Leu Thr Ala Ala Ala Arg Leu Leu Gly Thr Lys Asn Ala Ser1 5 10
15Cys Leu Val Leu Ala Ala Arg His Ala Ser 20 252435PRTHomo sapiens
24Gln Ser Trp Ser Arg Val Tyr Cys Ser Leu Ala Lys Arg Gly His Phe1
5 10 15Asn Arg Ile Ser His Gly Leu Gln Gly Leu Ser Ala Val Pro Leu
Arg 20 25 30Thr Tyr Ala 352534PRTHomo sapiens 25Gly Ala Leu Val Ile
Arg Gly Ile Arg Asn Phe Asn Leu Glu Asn Arg1 5 10 15Ala Glu Arg Glu
Ile Ser Lys Met Lys Pro Ser Val Ala Pro Arg His 20 25 30Pro
Ser26130PRTHomo sapiens 26Ser Gly Thr Leu Gly His Pro Gly Ser Leu
Asp Glu Thr Thr Tyr Glu1 5 10 15Arg Leu Ala Glu Glu Thr Leu Asp Ser
Leu Ala Glu Phe Phe Glu Asp 20 25 30Leu Ala Asp Lys Pro Tyr Thr Phe
Glu Asp Tyr Asp Val Ser Phe Gly 35 40 45Ser Gly Val Leu Thr Val Lys
Leu Gly Gly Asp Leu Gly Thr Tyr Val 50 55 60Ile Asn Lys Gln Thr Pro
Asn Lys Gln Ile Trp Leu Ser Ser Pro Ser65 70 75 80Ser Gly Pro Lys
Arg Tyr Asp Trp Thr Gly Lys Asn Trp Val Tyr Ser 85 90 95His Asp Gly
Val Ser Leu His Glu Leu Leu Ala Ala Glu Leu Thr Lys 100 105 110Ala
Leu Lys Thr Lys Leu Asp Leu Ser Ser Leu Ala Tyr Ser Gly Lys 115 120
125Asp Ala 130279PRTHuman immunodeficiency virus type 2 27Arg Lys
Lys Arg Arg Gln Arg Arg Arg1 528170PRTArtificial SequenceTAT
MTS(cs) 81-210 FRA 28Met Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly
Ser Asp Pro Ala Leu1 5 10 15Leu Thr Ala Ala Ala Arg Leu Leu Gly Thr
Lys Asn Ala Ser Cys Leu 20 25 30Val Leu Ala Ala Arg His Ala Ser Ser
Gly Thr Leu Gly His Pro Gly 35 40 45Ser Leu Asp Glu Thr Thr Tyr Glu
Arg Leu Ala Glu Glu Thr Leu Asp 50 55 60Ser Leu Ala Glu Phe Phe Glu
Asp Leu Ala Asp Lys Pro Tyr Thr Phe65 70 75 80Glu Asp Tyr Asp Val
Ser Phe Gly Ser Gly Val Leu Thr Val Lys Leu 85 90 95Gly Gly Asp Leu
Gly Thr Tyr Val Ile Asn Lys Gln Thr Pro Asn Lys 100 105 110Gln Ile
Trp Leu Ser Ser Pro Ser Ser Gly Pro Lys Arg Tyr Asp Trp 115 120
125Thr Gly Lys Asn Trp Val Tyr Ser His Asp Gly Val Ser Leu His Glu
130 135 140Leu Leu Ala Ala Glu Leu Thr Lys Ala Leu Lys Thr Lys Leu
Asp Leu145 150 155 160Ser Ser Leu Ala Tyr Ser Gly Lys Asp Ala 165
17029223PRTArtificial SequenceTAT MTS(fra)81-210 FRA 29Met Arg Lys
Lys Arg Arg Gln Arg Arg Arg Gly Ser Asp Pro Trp Thr1 5 10 15Leu Gly
Arg Arg Ala Val Ala Gly Leu Leu Ala Ser Pro Ser Pro Ala 20 25 30Gln
Ala Gln Thr Leu Thr Arg Val Pro Arg Pro Ala Glu Leu Ala Pro 35 40
45Leu Cys Gly Arg Arg Gly Leu Arg Thr Asp Ile Asp Ala Thr Cys Thr
50 55 60Pro Arg Arg Ala Ser Ser Asn Gln Arg Gly Leu Asn Gln Ile Trp
Asn65 70 75 80Val Lys Lys Gln Ser Val Tyr Leu Met Asn Leu Arg Lys
Ser Gly Thr 85 90 95Leu Gly His Pro Gly Ser Leu Asp Glu Thr Thr Tyr
Glu Arg Leu Ala 100 105 110Glu Glu Thr Leu Asp Ser Leu Ala Glu Phe
Phe Glu Asp Leu Ala Asp 115 120 125Lys Pro Tyr Thr Phe Glu Asp Tyr
Asp Val Ser Phe Gly Ser Gly Val 130 135 140Leu Thr Val Lys Leu Gly
Gly Asp Leu Gly Thr Tyr Val Ile Asn Lys145 150 155 160Gln Thr Pro
Asn Lys Gln Ile Trp Leu Ser Ser Pro Ser Ser Gly Pro 165 170 175Lys
Arg Tyr Asp Trp Thr Gly Lys Asn Trp Val Tyr Ser His Asp Gly 180 185
190Val Ser Leu His Glu Leu Leu Ala Ala Glu Leu Thr Lys Ala Leu Lys
195 200 205Thr Lys Leu Asp Leu Ser Ser Leu Ala Tyr Ser Gly Lys Asp
Ala 210 215 22030179PRTArtificial SequenceTAT MTS(lad) 81-210 FRA
30Met Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Ser Asp Pro Gln Ser1
5 10 15Trp Ser Arg Val Tyr Cys Ser Leu Ala Lys Arg Gly His Phe Asn
Arg 20 25 30Ile Ser His Gly Leu Gln Gly Leu Ser Ala Val Pro Leu Arg
Thr Tyr 35 40 45Ala Ser Gly Thr Leu Gly His Pro Gly Ser Leu Asp Glu
Thr Thr Tyr 50 55 60Glu Arg Leu Ala Glu Glu Thr Leu Asp Ser Leu Ala
Glu Phe Phe Glu65 70 75 80Asp Leu Ala Asp Lys Pro Tyr Thr Phe Glu
Asp Tyr Asp Val Ser Phe 85 90 95Gly Ser Gly Val Leu Thr Val Lys Leu
Gly Gly Asp Leu Gly Thr Tyr 100 105 110Val Ile Asn Lys Gln Thr Pro
Asn Lys Gln Ile Trp Leu Ser Ser Pro 115 120 125Ser Ser Gly Pro Lys
Arg Tyr Asp Trp Thr Gly Lys Asn Trp Val Tyr 130 135 140Ser His Asp
Gly Val Ser Leu His Glu Leu Leu Ala Ala Glu Leu Thr145 150 155
160Lys Ala Leu Lys Thr Lys Leu Asp Leu Ser Ser Leu Ala Tyr Ser Gly
165 170 175Lys Asp Ala31178PRTArtificial SequenceTAT MTS(orf)
81-210 FRA 31Met Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Ser Asp
Pro Gly Ala1 5 10 15Leu Val Ile Arg Gly Ile Arg Asn Phe Asn Leu Glu
Asn Arg Ala Glu 20 25 30Arg Glu Ile Ser Lys Met Lys Pro Ser Val Ala
Pro Arg His Pro Ser 35 40 45Ser Gly Thr Leu Gly His Pro Gly Ser Leu
Asp Glu Thr Thr Tyr Glu 50 55 60Arg Leu Ala Glu Glu Thr Leu Asp Ser
Leu Ala Glu Phe Phe Glu Asp65 70 75 80Leu Ala Asp Lys Pro
Tyr Thr Phe Glu Asp Tyr Asp Val Ser Phe Gly 85 90 95Ser Gly Val Leu
Thr Val Lys Leu Gly Gly Asp Leu Gly Thr Tyr Val 100 105 110Ile Asn
Lys Gln Thr Pro Asn Lys Gln Ile Trp Leu Ser Ser Pro Ser 115 120
125Ser Gly Pro Lys Arg Tyr Asp Trp Thr Gly Lys Asn Trp Val Tyr Ser
130 135 140His Asp Gly Val Ser Leu His Glu Leu Leu Ala Ala Glu Leu
Thr Lys145 150 155 160Ala Leu Lys Thr Lys Leu Asp Leu Ser Ser Leu
Ala Tyr Ser Gly Lys 165 170 175Asp Ala324PRTArtificial
Sequencepolypeptide linker 32Gly Ser Asp Pro133166PRTArtificial
SequenceTAT MTS(cs) 81-210 FRA delta linker 33Met Arg Lys Lys Arg
Arg Gln Arg Arg Arg Ala Leu Leu Thr Ala Ala1 5 10 15Ala Arg Leu Leu
Gly Thr Lys Asn Ala Ser Cys Leu Val Leu Ala Ala 20 25 30Arg His Ala
Ser Ser Gly Thr Leu Gly His Pro Gly Ser Leu Asp Glu 35 40 45Thr Thr
Tyr Glu Arg Leu Ala Glu Glu Thr Leu Asp Ser Leu Ala Glu 50 55 60Phe
Phe Glu Asp Leu Ala Asp Lys Pro Tyr Thr Phe Glu Asp Tyr Asp65 70 75
80Val Ser Phe Gly Ser Gly Val Leu Thr Val Lys Leu Gly Gly Asp Leu
85 90 95Gly Thr Tyr Val Ile Asn Lys Gln Thr Pro Asn Lys Gln Ile Trp
Leu 100 105 110Ser Ser Pro Ser Ser Gly Pro Lys Arg Tyr Asp Trp Thr
Gly Lys Asn 115 120 125Trp Val Tyr Ser His Asp Gly Val Ser Leu His
Glu Leu Leu Ala Ala 130 135 140Glu Leu Thr Lys Ala Leu Lys Thr Lys
Leu Asp Leu Ser Ser Leu Ala145 150 155 160Tyr Ser Gly Lys Asp Ala
16534219PRTArtificial SequenceTAT MTS(fra)81-210 FRA delta linker
34Met Arg Lys Lys Arg Arg Gln Arg Arg Arg Trp Thr Leu Gly Arg Arg1
5 10 15Ala Val Ala Gly Leu Leu Ala Ser Pro Ser Pro Ala Gln Ala Gln
Thr 20 25 30Leu Thr Arg Val Pro Arg Pro Ala Glu Leu Ala Pro Leu Cys
Gly Arg 35 40 45Arg Gly Leu Arg Thr Asp Ile Asp Ala Thr Cys Thr Pro
Arg Arg Ala 50 55 60Ser Ser Asn Gln Arg Gly Leu Asn Gln Ile Trp Asn
Val Lys Lys Gln65 70 75 80Ser Val Tyr Leu Met Asn Leu Arg Lys Ser
Gly Thr Leu Gly His Pro 85 90 95Gly Ser Leu Asp Glu Thr Thr Tyr Glu
Arg Leu Ala Glu Glu Thr Leu 100 105 110Asp Ser Leu Ala Glu Phe Phe
Glu Asp Leu Ala Asp Lys Pro Tyr Thr 115 120 125Phe Glu Asp Tyr Asp
Val Ser Phe Gly Ser Gly Val Leu Thr Val Lys 130 135 140Leu Gly Gly
Asp Leu Gly Thr Tyr Val Ile Asn Lys Gln Thr Pro Asn145 150 155
160Lys Gln Ile Trp Leu Ser Ser Pro Ser Ser Gly Pro Lys Arg Tyr Asp
165 170 175Trp Thr Gly Lys Asn Trp Val Tyr Ser His Asp Gly Val Ser
Leu His 180 185 190Glu Leu Leu Ala Ala Glu Leu Thr Lys Ala Leu Lys
Thr Lys Leu Asp 195 200 205Leu Ser Ser Leu Ala Tyr Ser Gly Lys Asp
Ala 210 21535175PRTArtificial SequenceTAT MTS(lad) 81-210 FRA delta
linker 35Met Arg Lys Lys Arg Arg Gln Arg Arg Arg Gln Ser Trp Ser
Arg Val1 5 10 15Tyr Cys Ser Leu Ala Lys Arg Gly His Phe Asn Arg Ile
Ser His Gly 20 25 30Leu Gln Gly Leu Ser Ala Val Pro Leu Arg Thr Tyr
Ala Ser Gly Thr 35 40 45Leu Gly His Pro Gly Ser Leu Asp Glu Thr Thr
Tyr Glu Arg Leu Ala 50 55 60Glu Glu Thr Leu Asp Ser Leu Ala Glu Phe
Phe Glu Asp Leu Ala Asp65 70 75 80Lys Pro Tyr Thr Phe Glu Asp Tyr
Asp Val Ser Phe Gly Ser Gly Val 85 90 95Leu Thr Val Lys Leu Gly Gly
Asp Leu Gly Thr Tyr Val Ile Asn Lys 100 105 110Gln Thr Pro Asn Lys
Gln Ile Trp Leu Ser Ser Pro Ser Ser Gly Pro 115 120 125Lys Arg Tyr
Asp Trp Thr Gly Lys Asn Trp Val Tyr Ser His Asp Gly 130 135 140Val
Ser Leu His Glu Leu Leu Ala Ala Glu Leu Thr Lys Ala Leu Lys145 150
155 160Thr Lys Leu Asp Leu Ser Ser Leu Ala Tyr Ser Gly Lys Asp Ala
165 170 17536174PRTArtificial SequenceTAT MTS(orf) 81-210 FRA delta
linker 36Met Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Ala Leu Val
Ile Arg1 5 10 15Gly Ile Arg Asn Phe Asn Leu Glu Asn Arg Ala Glu Arg
Glu Ile Ser 20 25 30Lys Met Lys Pro Ser Val Ala Pro Arg His Pro Ser
Ser Gly Thr Leu 35 40 45Gly His Pro Gly Ser Leu Asp Glu Thr Thr Tyr
Glu Arg Leu Ala Glu 50 55 60Glu Thr Leu Asp Ser Leu Ala Glu Phe Phe
Glu Asp Leu Ala Asp Lys65 70 75 80Pro Tyr Thr Phe Glu Asp Tyr Asp
Val Ser Phe Gly Ser Gly Val Leu 85 90 95Thr Val Lys Leu Gly Gly Asp
Leu Gly Thr Tyr Val Ile Asn Lys Gln 100 105 110Thr Pro Asn Lys Gln
Ile Trp Leu Ser Ser Pro Ser Ser Gly Pro Lys 115 120 125Arg Tyr Asp
Trp Thr Gly Lys Asn Trp Val Tyr Ser His Asp Gly Val 130 135 140Ser
Leu His Glu Leu Leu Ala Ala Glu Leu Thr Lys Ala Leu Lys Thr145 150
155 160Lys Leu Asp Leu Ser Ser Leu Ala Tyr Ser Gly Lys Asp Ala 165
17037105DNAHomo sapiens 37ctgtttaacc tgcgcattct gctgaacaat
gcggccttcc gtaacggcca taattttatg 60gtccgcaact tccgttgcgg tcagccgctg
caaaataaag tgcag 1053835PRTHomo sapiens 38Leu Phe Asn Leu Arg Ile
Leu Leu Asn Asn Ala Ala Phe Arg Asn Gly1 5 10 15His Asn Phe Met Val
Arg Asn Phe Arg Cys Gly Gln Pro Leu Gln Asn 20 25 30Lys Val Gln
3539318PRTHomo sapiens 39Leu Lys Gly Arg Asp Leu Leu Thr Leu Arg
Asn Phe Thr Gly Glu Glu1 5 10 15Ile Lys Tyr Met Leu Trp Leu Ser Ala
Asp Leu Lys Phe Arg Ile Lys 20 25 30Gln Lys Gly Glu Tyr Leu Pro Leu
Leu Gln Gly Lys Ser Leu Gly Met 35 40 45Ile Phe Glu Lys Arg Ser Thr
Arg Thr Arg Leu Ser Thr Glu Thr Gly 50 55 60Phe Ala Leu Leu Gly Gly
His Pro Cys Phe Leu Thr Thr Gln Asp Ile65 70 75 80His Leu Gly Val
Asn Glu Ser Leu Thr Asp Thr Ala Arg Val Leu Ser 85 90 95Ser Met Ala
Asp Ala Val Leu Ala Arg Val Tyr Lys Gln Ser Asp Leu 100 105 110Asp
Thr Leu Ala Lys Glu Ala Ser Ile Pro Ile Ile Asn Gly Leu Ser 115 120
125Asp Leu Tyr His Pro Ile Gln Ile Leu Ala Asp Tyr Leu Thr Leu Gln
130 135 140Glu His Tyr Ser Ser Leu Lys Gly Leu Thr Leu Ser Trp Ile
Gly Asp145 150 155 160Gly Asn Asn Ile Leu His Ser Ile Met Met Ser
Ala Ala Lys Phe Gly 165 170 175Met His Leu Gln Ala Ala Thr Pro Lys
Gly Tyr Glu Pro Asp Ala Ser 180 185 190Val Thr Lys Leu Ala Glu Gln
Tyr Ala Lys Glu Asn Gly Thr Lys Leu 195 200 205Leu Leu Thr Asn Asp
Pro Leu Glu Ala Ala His Gly Gly Asn Val Leu 210 215 220Ile Thr Asp
Thr Trp Ile Ser Met Gly Gln Glu Glu Glu Lys Lys Lys225 230 235
240Arg Leu Gln Ala Phe Gln Gly Tyr Gln Val Thr Met Lys Thr Ala Lys
245 250 255Val Ala Ala Ser Asp Trp Thr Phe Leu His Cys Leu Pro Arg
Lys Pro 260 265 270Glu Glu Val Asp Asp Glu Val Phe Tyr Ser Pro Arg
Ser Leu Val Phe 275 280 285Pro Glu Ala Glu Asn Arg Lys Trp Thr Ile
Met Ala Val Met Val Ser 290 295 300Leu Leu Thr Asp Tyr Ser Pro Gln
Leu Gln Lys Pro Lys Phe305 310 315401170DNAArtificial
SequenceHis-TAT-MTS(otc)-OTC delta inker 40catatgggct catcgcatca
tcatcatcat cactcatcag gtctggttcc gcgtggctcg 60cacatgtatg gtcgcaaaaa
acgtcgtcaa cgtcgccgtc tgtttaacct gcgcattctg 120ctgaacaatg
cggccttccg taacggccat aattttatgg tccgcaactt ccgttgcggt
180cagccgctgc aaaataaagt gcagctgaaa ggccgcgatc tgctgaccct
gcgtaacttc 240acgggtgaag aaatcaaata catgctgtgg ctgagcgcag
acctgaaatt ccgcatcaaa 300caaaaaggcg aatacctgcc gctgctgcag
ggcaaatctc tgggtatgat ttttgaaaaa 360cgtagtaccc gcacgcgtct
gtccaccgaa acgggctttg ccctgctggg cggtcatccg 420tgtttcctga
ccacgcaaga tatccacctg ggtgtgaacg aaagtctgac cgatacggca
480cgcgttctga gctctatggc agacgctgtg ctggctcgtg tttataaaca
gtccgatctg 540gacaccctgg cgaaagaagc ctcaattccg attatcaatg
gcctgtcgga tctgtaccat 600ccgattcaaa tcctggcgga ctatctgacc
ctgcaggaac actacagttc cctgaaaggt 660ctgaccctga gttggatcgg
cgatggtaac aatattctgc atagcatcat gatgtctgca 720gctaaatttg
gcatgcacct gcaagcggcc accccgaaag gttatgaacc ggatgccagc
780gttacgaaac tggcagaaca gtacgctaaa gaaaacggta ccaaactgct
gctgacgaat 840gatccgctgg aagcagctca tggcggtaac gtcctgatta
ccgacacgtg gatctctatg 900ggccaggaag aagaaaagaa aaaacgtctg
caggcgtttc aaggttatca ggttaccatg 960aaaacggcca aagtcgcggc
cagcgattgg accttcctgc actgcctgcc gcgtaaaccg 1020gaagaagtcg
atgacgaagt gttttactca ccgcgctcgc tggtgttccc ggaagcagaa
1080aatcgtaaat ggaccatcat ggctgttatg gtgtccctgc tgaccgacta
ttccccgcaa 1140ctgcaaaaac cgaaattcta atgaaagctt
1170411155DNAArtificial SequenceHis-TAT-MTS(cs)-OTC 41catatgggct
catctcatca tcatcatcat cattcgtcag gtctggtccc gcgtggctct 60cacatgcgta
aaaaacgtcg tcagcgtcgt cgtggcagtg atccggcact gctgaccgca
120gcagcacgtc tgctgggtac gaaaaacgct agctgcctgg tgctggctgc
gcgtcatgcg 180tctgaatttc tgaaaggccg tgatctgctg accctgcgca
acttcacggg tgaagaaatc 240aaatacatgc tgtggctgag tgccgacctg
aaatttcgta tcaaacaaaa aggcgaatac 300ctgccgctgc tgcagggcaa
atccctgggt atgattttcg aaaaacgcag tacccgtacg 360cgcctgtcca
ccgaaacggg ctttgcactg ctgggcggtc atccgtgttt cctgaccacg
420caagatatcc acctgggtgt gaacgaatca ctgaccgata cggctcgtgt
tctgagctct 480atggcagacg cagtgctggc acgtgtttat aaacagtcgg
atctggacac cctggctaaa 540gaagcgtcaa ttccgattat caatggcctg
tcggatctgt accatccgat tcaaatcctg 600gcggactatc tgaccctgca
ggaacactac agttccctga aaggtctgac cctgagctgg 660atcggcgatg
gtaacaatat tctgcatagc atcatgatgt ctgccgcaaa atttggcatg
720cacctgcaag ctgcgacccc gaaaggttat gaaccggacg ccagcgtcac
gaaactggcc 780gaacagtacg caaaagaaaa cggtaccaaa ctgctgctga
cgaatgatcc gctggaagcc 840gcacatggcg gtaacgttct gattaccgac
acgtggatca gcatgggcca ggaagaagaa 900aagaaaaaac gtctgcaggc
ctttcaaggt tatcaggtta ccatgaaaac ggcaaaagtc 960gctgcgtctg
attggacctt cctgcactgc ctgccgcgca aaccggaaga agtcgatgac
1020gaagtgtttt actcaccgcg ttcgctggtt ttcccggaag cggaaaatcg
caaatggacc 1080attatggctg tgatggtctc tctgctgacg gactactcgc
cgcaactgca aaaaccgaaa 1140ttctaatgaa agctt 1155421179DNAArtificial
SequenceHis-TAT-MTS(orf)-OTC 42catatgggtt catcacatca tcatcatcat
cattcatcag gtctggtccc gcgtggttca 60cacatgcgta aaaaacgtcg tcagcgtcgt
cgtggcagtg atccgggtgc gctggtcatt 120cgtggcatcc gcaactttaa
tctggaaaac cgtgcggaac gcgaaattag taaaatgaaa 180ccgtccgtgg
caccgcgtca tccgtctgaa tttctgaaag gccgtgatct gctgaccctg
240cgcaacttca cgggtgaaga aatcaaatac atgctgtggc tgagtgcaga
cctgaaattc 300cgtatcaaac aaaagggtga atacctgccg ctgctgcagg
gcaaatccct gggtatgatt 360ttcgaaaaac gctcaacccg tacgcgcctg
tcgaccgaaa cgggctttgc cctgctgggc 420ggtcatccgt gcttcctgac
cacgcaagat atccacctgg gtgtgaacga atcactgacc 480gatacggcac
gtgttctgag ctctatggca gacgcagtgc tggctcgtgt ttataaacag
540tcggatctgg acaccctggc aaaagaagct agcattccga ttatcaatgg
cctgtctgat 600ctgtaccatc cgattcaaat cctggcggac tatctgaccc
tgcaggaaca ctacagttcc 660ctgaaaggtc tgaccctgag ctggatcggc
gatggtaaca atattctgca tagcatcatg 720atgtctgcgg ccaaattcgg
catgcacctg caagcagcta ccccgaaagg ttatgaaccg 780gacgcctccg
ttacgaaact ggcggaacag tacgccaaag aaaacggcac caaactgctg
840ctgacgaatg atccgctgga agcggcccat ggcggtaacg tcctgattac
cgacacgtgg 900atcagcatgg gccaggaaga agaaaagaaa aaacgtctgc
aggcatttca aggttatcag 960gttaccatga aaacggctaa agtcgcagct
tctgattgga ccttcctgca ctgtctgccg 1020cgcaaaccgg aagaagtcga
tgacgaagtg ttttactcac cgcgttcgct ggtgttcccg 1080gaagcggaaa
atcgcaaatg gaccattatg gctgtgatgg tgtcgctgct gacggactac
1140tcgccgcaac tgcaaaaacc gaaattctaa tgaaagctt
1179431182DNAArtificial SequenceHis-TAT-MTS(lad)-OTC 43catatgggta
gttcacatca tcatcatcat cactcgtcgg gtctggtgcc gcgtggctca 60cacatgcgta
aaaaacgtcg tcagcgtcgt cgtggctcag atccgcaatc atggtcgcgc
120gtctattgct cgctggcgaa acgtggtcat tttaaccgca ttagccacgg
cctgcagggt 180ctgtctgcag tgccgctgcg tacctacgct gaatttctga
aaggccgtga tctgctgacc 240ctgcgcaact tcacgggtga agaaatcaaa
tacatgctgt ggctgagcgc agacctgaaa 300tttcgtatca aacaaaaagg
cgaatacctg ccgctgctgc agggcaaatc tctgggtatg 360attttcgaaa
aacgctcaac ccgtacgcgc ctgtcgaccg aaacgggctt tgccctgctg
420ggcggtcatc cgtgtttcct gaccacgcag gatatccacc tgggtgtgaa
cgaaagtctg 480accgatacgg cacgtgttct gagctctatg gcagacgcag
tgctggctcg tgtttataaa 540cagtccgatc tggacaccct ggcaaaagaa
gctagtattc cgattatcaa tggcctgtcc 600gatctgtacc atccgattca
aatcctggcg gactatctga ccctgcagga acactacagt 660tccctgaaag
gtctgacgct gagctggatc ggcgatggta acaatattct gcatagtatc
720atgatgtccg cggccaaatt cggcatgcac ctgcaagcag ctaccccgaa
aggttatgaa 780ccggacgcct ctgttacgaa actggcggaa cagtacgcca
aagaaaacgg taccaaactg 840ctgctgacga atgatccgct ggaagcggcc
catggcggta acgtcctgat taccgacacg 900tggatcagta tgggccagga
agaagaaaag aaaaaacgtc tgcaggcgtt tcaaggttat 960caggttacca
tgaaaacggc caaagtcgca gctagcgatt ggaccttcct gcactgcctg
1020ccgcgcaaac cggaagaagt cgatgacgaa gtgttttata gcccgcgttc
tctggtgttc 1080ccggaagcgg aaaatcgcaa atggaccatc atggccgtta
tggtgtcgct gctgaccgat 1140tactccccgc aactgcaaaa accgaaattc
taatgaaagc tt 118244385PRTArtificial SequenceHis-TAT-MTS(otc)-OTC
delta linker 44Met Gly Ser Ser His His His His His His Ser Ser Gly
Leu Val Pro1 5 10 15Arg Gly Ser His Met Tyr Gly Arg Lys Lys Arg Arg
Gln Arg Arg Arg 20 25 30Leu Phe Asn Leu Arg Ile Leu Leu Asn Asn Ala
Ala Phe Arg Asn Gly 35 40 45His Asn Phe Met Val Arg Asn Phe Arg Cys
Gly Gln Pro Leu Gln Asn 50 55 60Lys Val Gln Leu Lys Gly Arg Asp Leu
Leu Thr Leu Arg Asn Phe Thr65 70 75 80Gly Glu Glu Ile Lys Tyr Met
Leu Trp Leu Ser Ala Asp Leu Lys Phe 85 90 95Arg Ile Lys Gln Lys Gly
Glu Tyr Leu Pro Leu Leu Gln Gly Lys Ser 100 105 110Leu Gly Met Ile
Phe Glu Lys Arg Ser Thr Arg Thr Arg Leu Ser Thr 115 120 125Glu Thr
Gly Phe Ala Leu Leu Gly Gly His Pro Cys Phe Leu Thr Thr 130 135
140Gln Asp Ile His Leu Gly Val Asn Glu Ser Leu Thr Asp Thr Ala
Arg145 150 155 160Val Leu Ser Ser Met Ala Asp Ala Val Leu Ala Arg
Val Tyr Lys Gln 165 170 175Ser Asp Leu Asp Thr Leu Ala Lys Glu Ala
Ser Ile Pro Ile Ile Asn 180 185 190Gly Leu Ser Asp Leu Tyr His Pro
Ile Gln Ile Leu Ala Asp Tyr Leu 195 200 205Thr Leu Gln Glu His Tyr
Ser Ser Leu Lys Gly Leu Thr Leu Ser Trp 210 215 220Ile Gly Asp Gly
Asn Asn Ile Leu His Ser Ile Met Met Ser Ala Ala225 230 235 240Lys
Phe Gly Met His Leu Gln Ala Ala Thr Pro Lys Gly Tyr Glu Pro 245 250
255Asp Ala Ser Val Thr Lys Leu Ala Glu Gln Tyr Ala Lys Glu Asn Gly
260 265 270Thr Lys Leu Leu Leu Thr Asn Asp Pro Leu Glu Ala Ala His
Gly Gly 275 280 285Asn Val Leu Ile Thr Asp Thr Trp Ile Ser Met Gly
Gln Glu Glu Glu 290 295 300Lys Lys Lys Arg Leu Gln Ala Phe Gln Gly
Tyr Gln Val Thr Met Lys305 310 315 320Thr Ala Lys Val Ala Ala Ser
Asp Trp Thr Phe Leu His Cys Leu Pro 325 330 335Arg Lys Pro Glu Glu
Val Asp Asp Glu Val Phe Tyr Ser Pro Arg Ser 340 345 350Leu Val Phe
Pro Glu Ala Glu Asn Arg Lys Trp Thr Ile Met Ala Val 355 360 365Met
Val Ser Leu Leu Thr Asp Tyr Ser Pro Gln Leu Gln Lys Pro Lys 370
375
380Phe38545380PRTArtificial SequenceHis-TAT-MTS(cs)-OTC 45Met Gly
Ser Ser His His His His His His Ser Ser Gly Leu Val Pro1 5 10 15Arg
Gly Ser His Met Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Ser 20 25
30Asp Pro Ala Leu Leu Thr Ala Ala Ala Arg Leu Leu Gly Thr Lys Asn
35 40 45Ala Ser Cys Leu Val Leu Ala Ala Arg His Ala Ser Glu Phe Leu
Lys 50 55 60Gly Arg Asp Leu Leu Thr Leu Arg Asn Phe Thr Gly Glu Glu
Ile Lys65 70 75 80Tyr Met Leu Trp Leu Ser Ala Asp Leu Lys Phe Arg
Ile Lys Gln Lys 85 90 95Gly Glu Tyr Leu Pro Leu Leu Gln Gly Lys Ser
Leu Gly Met Ile Phe 100 105 110Glu Lys Arg Ser Thr Arg Thr Arg Leu
Ser Thr Glu Thr Gly Phe Ala 115 120 125Leu Leu Gly Gly His Pro Cys
Phe Leu Thr Thr Gln Asp Ile His Leu 130 135 140Gly Val Asn Glu Ser
Leu Thr Asp Thr Ala Arg Val Leu Ser Ser Met145 150 155 160Ala Asp
Ala Val Leu Ala Arg Val Tyr Lys Gln Ser Asp Leu Asp Thr 165 170
175Leu Ala Lys Glu Ala Ser Ile Pro Ile Ile Asn Gly Leu Ser Asp Leu
180 185 190Tyr His Pro Ile Gln Ile Leu Ala Asp Tyr Leu Thr Leu Gln
Glu His 195 200 205Tyr Ser Ser Leu Lys Gly Leu Thr Leu Ser Trp Ile
Gly Asp Gly Asn 210 215 220Asn Ile Leu His Ser Ile Met Met Ser Ala
Ala Lys Phe Gly Met His225 230 235 240Leu Gln Ala Ala Thr Pro Lys
Gly Tyr Glu Pro Asp Ala Ser Val Thr 245 250 255Lys Leu Ala Glu Gln
Tyr Ala Lys Glu Asn Gly Thr Lys Leu Leu Leu 260 265 270Thr Asn Asp
Pro Leu Glu Ala Ala His Gly Gly Asn Val Leu Ile Thr 275 280 285Asp
Thr Trp Ile Ser Met Gly Gln Glu Glu Glu Lys Lys Lys Arg Leu 290 295
300Gln Ala Phe Gln Gly Tyr Gln Val Thr Met Lys Thr Ala Lys Val
Ala305 310 315 320Ala Ser Asp Trp Thr Phe Leu His Cys Leu Pro Arg
Lys Pro Glu Glu 325 330 335Val Asp Asp Glu Val Phe Tyr Ser Pro Arg
Ser Leu Val Phe Pro Glu 340 345 350Ala Glu Asn Arg Lys Trp Thr Ile
Met Ala Val Met Val Ser Leu Leu 355 360 365Thr Asp Tyr Ser Pro Gln
Leu Gln Lys Pro Lys Phe 370 375 38046388PRTArtificial
SequenceHis-TAT-MTS(orf)-OTC 46Met Gly Ser Ser His His His His His
His Ser Ser Gly Leu Val Pro1 5 10 15Arg Gly Ser His Met Arg Lys Lys
Arg Arg Gln Arg Arg Arg Gly Ser 20 25 30Asp Pro Gly Ala Leu Val Ile
Arg Gly Ile Arg Asn Phe Asn Leu Glu 35 40 45Asn Arg Ala Glu Arg Glu
Ile Ser Lys Met Lys Pro Ser Val Ala Pro 50 55 60Arg His Pro Ser Glu
Phe Leu Lys Gly Arg Asp Leu Leu Thr Leu Arg65 70 75 80Asn Phe Thr
Gly Glu Glu Ile Lys Tyr Met Leu Trp Leu Ser Ala Asp 85 90 95Leu Lys
Phe Arg Ile Lys Gln Lys Gly Glu Tyr Leu Pro Leu Leu Gln 100 105
110Gly Lys Ser Leu Gly Met Ile Phe Glu Lys Arg Ser Thr Arg Thr Arg
115 120 125Leu Ser Thr Glu Thr Gly Phe Ala Leu Leu Gly Gly His Pro
Cys Phe 130 135 140Leu Thr Thr Gln Asp Ile His Leu Gly Val Asn Glu
Ser Leu Thr Asp145 150 155 160Thr Ala Arg Val Leu Ser Ser Met Ala
Asp Ala Val Leu Ala Arg Val 165 170 175Tyr Lys Gln Ser Asp Leu Asp
Thr Leu Ala Lys Glu Ala Ser Ile Pro 180 185 190Ile Ile Asn Gly Leu
Ser Asp Leu Tyr His Pro Ile Gln Ile Leu Ala 195 200 205Asp Tyr Leu
Thr Leu Gln Glu His Tyr Ser Ser Leu Lys Gly Leu Thr 210 215 220Leu
Ser Trp Ile Gly Asp Gly Asn Asn Ile Leu His Ser Ile Met Met225 230
235 240Ser Ala Ala Lys Phe Gly Met His Leu Gln Ala Ala Thr Pro Lys
Gly 245 250 255Tyr Glu Pro Asp Ala Ser Val Thr Lys Leu Ala Glu Gln
Tyr Ala Lys 260 265 270Glu Asn Gly Thr Lys Leu Leu Leu Thr Asn Asp
Pro Leu Glu Ala Ala 275 280 285His Gly Gly Asn Val Leu Ile Thr Asp
Thr Trp Ile Ser Met Gly Gln 290 295 300Glu Glu Glu Lys Lys Lys Arg
Leu Gln Ala Phe Gln Gly Tyr Gln Val305 310 315 320Thr Met Lys Thr
Ala Lys Val Ala Ala Ser Asp Trp Thr Phe Leu His 325 330 335Cys Leu
Pro Arg Lys Pro Glu Glu Val Asp Asp Glu Val Phe Tyr Ser 340 345
350Pro Arg Ser Leu Val Phe Pro Glu Ala Glu Asn Arg Lys Trp Thr Ile
355 360 365Met Ala Val Met Val Ser Leu Leu Thr Asp Tyr Ser Pro Gln
Leu Gln 370 375 380Lys Pro Lys Phe38547389PRTArtificial
SequenceHis-TAT-MTS(lad)-OTC 47Met Gly Ser Ser His His His His His
His Ser Ser Gly Leu Val Pro1 5 10 15Arg Gly Ser His Met Arg Lys Lys
Arg Arg Gln Arg Arg Arg Gly Ser 20 25 30Asp Pro Gln Ser Trp Ser Arg
Val Tyr Cys Ser Leu Ala Lys Arg Gly 35 40 45His Phe Asn Arg Ile Ser
His Gly Leu Gln Gly Leu Ser Ala Val Pro 50 55 60Leu Arg Thr Tyr Ala
Glu Phe Leu Lys Gly Arg Asp Leu Leu Thr Leu65 70 75 80Arg Asn Phe
Thr Gly Glu Glu Ile Lys Tyr Met Leu Trp Leu Ser Ala 85 90 95Asp Leu
Lys Phe Arg Ile Lys Gln Lys Gly Glu Tyr Leu Pro Leu Leu 100 105
110Gln Gly Lys Ser Leu Gly Met Ile Phe Glu Lys Arg Ser Thr Arg Thr
115 120 125Arg Leu Ser Thr Glu Thr Gly Phe Ala Leu Leu Gly Gly His
Pro Cys 130 135 140Phe Leu Thr Thr Gln Asp Ile His Leu Gly Val Asn
Glu Ser Leu Thr145 150 155 160Asp Thr Ala Arg Val Leu Ser Ser Met
Ala Asp Ala Val Leu Ala Arg 165 170 175Val Tyr Lys Gln Ser Asp Leu
Asp Thr Leu Ala Lys Glu Ala Ser Ile 180 185 190Pro Ile Ile Asn Gly
Leu Ser Asp Leu Tyr His Pro Ile Gln Ile Leu 195 200 205Ala Asp Tyr
Leu Thr Leu Gln Glu His Tyr Ser Ser Leu Lys Gly Leu 210 215 220Thr
Leu Ser Trp Ile Gly Asp Gly Asn Asn Ile Leu His Ser Ile Met225 230
235 240Met Ser Ala Ala Lys Phe Gly Met His Leu Gln Ala Ala Thr Pro
Lys 245 250 255Gly Tyr Glu Pro Asp Ala Ser Val Thr Lys Leu Ala Glu
Gln Tyr Ala 260 265 270Lys Glu Asn Gly Thr Lys Leu Leu Leu Thr Asn
Asp Pro Leu Glu Ala 275 280 285Ala His Gly Gly Asn Val Leu Ile Thr
Asp Thr Trp Ile Ser Met Gly 290 295 300Gln Glu Glu Glu Lys Lys Lys
Arg Leu Gln Ala Phe Gln Gly Tyr Gln305 310 315 320Val Thr Met Lys
Thr Ala Lys Val Ala Ala Ser Asp Trp Thr Phe Leu 325 330 335His Cys
Leu Pro Arg Lys Pro Glu Glu Val Asp Asp Glu Val Phe Tyr 340 345
350Ser Pro Arg Ser Leu Val Phe Pro Glu Ala Glu Asn Arg Lys Trp Thr
355 360 365Ile Met Ala Val Met Val Ser Leu Leu Thr Asp Tyr Ser Pro
Gln Leu 370 375 380Gln Lys Pro Lys Phe38548362PRTArtificial
SequenceTAT-MTS(otc)-OTC delta linker 48Arg Lys Lys Arg Arg Gln Arg
Arg Arg Leu Phe Asn Leu Arg Ile Leu1 5 10 15Leu Asn Asn Ala Ala Phe
Arg Asn Gly His Asn Phe Met Val Arg Asn 20 25 30Phe Arg Cys Gly Gln
Pro Leu Gln Asn Lys Val Gln Leu Lys Gly Arg 35 40 45Asp Leu Leu Thr
Leu Arg Asn Phe Thr Gly Glu Glu Ile Lys Tyr Met 50 55 60Leu Trp Leu
Ser Ala Asp Leu Lys Phe Arg Ile Lys Gln Lys Gly Glu65 70 75 80Tyr
Leu Pro Leu Leu Gln Gly Lys Ser Leu Gly Met Ile Phe Glu Lys 85 90
95Arg Ser Thr Arg Thr Arg Leu Ser Thr Glu Thr Gly Phe Ala Leu Leu
100 105 110Gly Gly His Pro Cys Phe Leu Thr Thr Gln Asp Ile His Leu
Gly Val 115 120 125Asn Glu Ser Leu Thr Asp Thr Ala Arg Val Leu Ser
Ser Met Ala Asp 130 135 140Ala Val Leu Ala Arg Val Tyr Lys Gln Ser
Asp Leu Asp Thr Leu Ala145 150 155 160Lys Glu Ala Ser Ile Pro Ile
Ile Asn Gly Leu Ser Asp Leu Tyr His 165 170 175Pro Ile Gln Ile Leu
Ala Asp Tyr Leu Thr Leu Gln Glu His Tyr Ser 180 185 190Ser Leu Lys
Gly Leu Thr Leu Ser Trp Ile Gly Asp Gly Asn Asn Ile 195 200 205Leu
His Ser Ile Met Met Ser Ala Ala Lys Phe Gly Met His Leu Gln 210 215
220Ala Ala Thr Pro Lys Gly Tyr Glu Pro Asp Ala Ser Val Thr Lys
Leu225 230 235 240Ala Glu Gln Tyr Ala Lys Glu Asn Gly Thr Lys Leu
Leu Leu Thr Asn 245 250 255Asp Pro Leu Glu Ala Ala His Gly Gly Asn
Val Leu Ile Thr Asp Thr 260 265 270Trp Ile Ser Met Gly Gln Glu Glu
Glu Lys Lys Lys Arg Leu Gln Ala 275 280 285Phe Gln Gly Tyr Gln Val
Thr Met Lys Thr Ala Lys Val Ala Ala Ser 290 295 300Asp Trp Thr Phe
Leu His Cys Leu Pro Arg Lys Pro Glu Glu Val Asp305 310 315 320Asp
Glu Val Phe Tyr Ser Pro Arg Ser Leu Val Phe Pro Glu Ala Glu 325 330
335Asn Arg Lys Trp Thr Ile Met Ala Val Met Val Ser Leu Leu Thr Asp
340 345 350Tyr Ser Pro Gln Leu Gln Lys Pro Lys Phe 355
36049360PRTArtificial SequenceTAT-MTS(cs)-OTC 49Met Arg Lys Lys Arg
Arg Gln Arg Arg Arg Gly Ser Asp Pro Ala Leu1 5 10 15Leu Thr Ala Ala
Ala Arg Leu Leu Gly Thr Lys Asn Ala Ser Cys Leu 20 25 30Val Leu Ala
Ala Arg His Ala Ser Glu Phe Leu Lys Gly Arg Asp Leu 35 40 45Leu Thr
Leu Arg Asn Phe Thr Gly Glu Glu Ile Lys Tyr Met Leu Trp 50 55 60Leu
Ser Ala Asp Leu Lys Phe Arg Ile Lys Gln Lys Gly Glu Tyr Leu65 70 75
80Pro Leu Leu Gln Gly Lys Ser Leu Gly Met Ile Phe Glu Lys Arg Ser
85 90 95Thr Arg Thr Arg Leu Ser Thr Glu Thr Gly Phe Ala Leu Leu Gly
Gly 100 105 110His Pro Cys Phe Leu Thr Thr Gln Asp Ile His Leu Gly
Val Asn Glu 115 120 125Ser Leu Thr Asp Thr Ala Arg Val Leu Ser Ser
Met Ala Asp Ala Val 130 135 140Leu Ala Arg Val Tyr Lys Gln Ser Asp
Leu Asp Thr Leu Ala Lys Glu145 150 155 160Ala Ser Ile Pro Ile Ile
Asn Gly Leu Ser Asp Leu Tyr His Pro Ile 165 170 175Gln Ile Leu Ala
Asp Tyr Leu Thr Leu Gln Glu His Tyr Ser Ser Leu 180 185 190Lys Gly
Leu Thr Leu Ser Trp Ile Gly Asp Gly Asn Asn Ile Leu His 195 200
205Ser Ile Met Met Ser Ala Ala Lys Phe Gly Met His Leu Gln Ala Ala
210 215 220Thr Pro Lys Gly Tyr Glu Pro Asp Ala Ser Val Thr Lys Leu
Ala Glu225 230 235 240Gln Tyr Ala Lys Glu Asn Gly Thr Lys Leu Leu
Leu Thr Asn Asp Pro 245 250 255Leu Glu Ala Ala His Gly Gly Asn Val
Leu Ile Thr Asp Thr Trp Ile 260 265 270Ser Met Gly Gln Glu Glu Glu
Lys Lys Lys Arg Leu Gln Ala Phe Gln 275 280 285Gly Tyr Gln Val Thr
Met Lys Thr Ala Lys Val Ala Ala Ser Asp Trp 290 295 300Thr Phe Leu
His Cys Leu Pro Arg Lys Pro Glu Glu Val Asp Asp Glu305 310 315
320Val Phe Tyr Ser Pro Arg Ser Leu Val Phe Pro Glu Ala Glu Asn Arg
325 330 335Lys Trp Thr Ile Met Ala Val Met Val Ser Leu Leu Thr Asp
Tyr Ser 340 345 350Pro Gln Leu Gln Lys Pro Lys Phe 355
36050368PRTArtificial SequenceTAT-MTS(orf)-OTC 50Met Arg Lys Lys
Arg Arg Gln Arg Arg Arg Gly Ser Asp Pro Gly Ala1 5 10 15Leu Val Ile
Arg Gly Ile Arg Asn Phe Asn Leu Glu Asn Arg Ala Glu 20 25 30Arg Glu
Ile Ser Lys Met Lys Pro Ser Val Ala Pro Arg His Pro Ser 35 40 45Glu
Phe Leu Lys Gly Arg Asp Leu Leu Thr Leu Arg Asn Phe Thr Gly 50 55
60Glu Glu Ile Lys Tyr Met Leu Trp Leu Ser Ala Asp Leu Lys Phe Arg65
70 75 80Ile Lys Gln Lys Gly Glu Tyr Leu Pro Leu Leu Gln Gly Lys Ser
Leu 85 90 95Gly Met Ile Phe Glu Lys Arg Ser Thr Arg Thr Arg Leu Ser
Thr Glu 100 105 110Thr Gly Phe Ala Leu Leu Gly Gly His Pro Cys Phe
Leu Thr Thr Gln 115 120 125Asp Ile His Leu Gly Val Asn Glu Ser Leu
Thr Asp Thr Ala Arg Val 130 135 140Leu Ser Ser Met Ala Asp Ala Val
Leu Ala Arg Val Tyr Lys Gln Ser145 150 155 160Asp Leu Asp Thr Leu
Ala Lys Glu Ala Ser Ile Pro Ile Ile Asn Gly 165 170 175Leu Ser Asp
Leu Tyr His Pro Ile Gln Ile Leu Ala Asp Tyr Leu Thr 180 185 190Leu
Gln Glu His Tyr Ser Ser Leu Lys Gly Leu Thr Leu Ser Trp Ile 195 200
205Gly Asp Gly Asn Asn Ile Leu His Ser Ile Met Met Ser Ala Ala Lys
210 215 220Phe Gly Met His Leu Gln Ala Ala Thr Pro Lys Gly Tyr Glu
Pro Asp225 230 235 240Ala Ser Val Thr Lys Leu Ala Glu Gln Tyr Ala
Lys Glu Asn Gly Thr 245 250 255Lys Leu Leu Leu Thr Asn Asp Pro Leu
Glu Ala Ala His Gly Gly Asn 260 265 270Val Leu Ile Thr Asp Thr Trp
Ile Ser Met Gly Gln Glu Glu Glu Lys 275 280 285Lys Lys Arg Leu Gln
Ala Phe Gln Gly Tyr Gln Val Thr Met Lys Thr 290 295 300Ala Lys Val
Ala Ala Ser Asp Trp Thr Phe Leu His Cys Leu Pro Arg305 310 315
320Lys Pro Glu Glu Val Asp Asp Glu Val Phe Tyr Ser Pro Arg Ser Leu
325 330 335Val Phe Pro Glu Ala Glu Asn Arg Lys Trp Thr Ile Met Ala
Val Met 340 345 350Val Ser Leu Leu Thr Asp Tyr Ser Pro Gln Leu Gln
Lys Pro Lys Phe 355 360 36551369PRTArtificial
SequenceTAT-MTS(lad)-OTC 51Met Arg Lys Lys Arg Arg Gln Arg Arg Arg
Gly Ser Asp Pro Gln Ser1 5 10 15Trp Ser Arg Val Tyr Cys Ser Leu Ala
Lys Arg Gly His Phe Asn Arg 20 25 30Ile Ser His Gly Leu Gln Gly Leu
Ser Ala Val Pro Leu Arg Thr Tyr 35 40 45Ala Glu Phe Leu Lys Gly Arg
Asp Leu Leu Thr Leu Arg Asn Phe Thr 50 55 60Gly Glu Glu Ile Lys Tyr
Met Leu Trp Leu Ser Ala Asp Leu Lys Phe65 70 75 80Arg Ile Lys Gln
Lys Gly Glu Tyr Leu Pro Leu Leu Gln Gly Lys Ser 85 90 95Leu Gly Met
Ile Phe Glu Lys Arg Ser Thr Arg Thr Arg Leu Ser Thr 100 105 110Glu
Thr Gly Phe Ala Leu Leu Gly Gly His Pro Cys Phe Leu Thr Thr 115 120
125Gln Asp Ile His Leu Gly Val Asn Glu Ser Leu Thr Asp Thr Ala Arg
130 135 140Val Leu Ser Ser Met Ala Asp Ala Val Leu Ala Arg Val Tyr
Lys Gln145 150 155 160Ser Asp Leu Asp Thr Leu Ala Lys Glu Ala Ser
Ile Pro Ile Ile Asn 165 170 175Gly Leu Ser Asp Leu Tyr His Pro Ile
Gln Ile Leu Ala Asp Tyr Leu 180 185
190Thr Leu Gln Glu His Tyr Ser Ser Leu Lys Gly Leu Thr Leu Ser Trp
195 200 205Ile Gly Asp Gly Asn Asn Ile Leu His Ser Ile Met Met Ser
Ala Ala 210 215 220Lys Phe Gly Met His Leu Gln Ala Ala Thr Pro Lys
Gly Tyr Glu Pro225 230 235 240Asp Ala Ser Val Thr Lys Leu Ala Glu
Gln Tyr Ala Lys Glu Asn Gly 245 250 255Thr Lys Leu Leu Leu Thr Asn
Asp Pro Leu Glu Ala Ala His Gly Gly 260 265 270Asn Val Leu Ile Thr
Asp Thr Trp Ile Ser Met Gly Gln Glu Glu Glu 275 280 285Lys Lys Lys
Arg Leu Gln Ala Phe Gln Gly Tyr Gln Val Thr Met Lys 290 295 300Thr
Ala Lys Val Ala Ala Ser Asp Trp Thr Phe Leu His Cys Leu Pro305 310
315 320Arg Lys Pro Glu Glu Val Asp Asp Glu Val Phe Tyr Ser Pro Arg
Ser 325 330 335Leu Val Phe Pro Glu Ala Glu Asn Arg Lys Trp Thr Ile
Met Ala Val 340 345 350Met Val Ser Leu Leu Thr Asp Tyr Ser Pro Gln
Leu Gln Lys Pro Lys 355 360 365Phe52356PRTArtificial
SequenceTAT-MTS(cs)-OTC delta linker 52Met Arg Lys Lys Arg Arg Gln
Arg Arg Arg Ala Leu Leu Thr Ala Ala1 5 10 15Ala Arg Leu Leu Gly Thr
Lys Asn Ala Ser Cys Leu Val Leu Ala Ala 20 25 30Arg His Ala Ser Glu
Phe Leu Lys Gly Arg Asp Leu Leu Thr Leu Arg 35 40 45Asn Phe Thr Gly
Glu Glu Ile Lys Tyr Met Leu Trp Leu Ser Ala Asp 50 55 60Leu Lys Phe
Arg Ile Lys Gln Lys Gly Glu Tyr Leu Pro Leu Leu Gln65 70 75 80Gly
Lys Ser Leu Gly Met Ile Phe Glu Lys Arg Ser Thr Arg Thr Arg 85 90
95Leu Ser Thr Glu Thr Gly Phe Ala Leu Leu Gly Gly His Pro Cys Phe
100 105 110Leu Thr Thr Gln Asp Ile His Leu Gly Val Asn Glu Ser Leu
Thr Asp 115 120 125Thr Ala Arg Val Leu Ser Ser Met Ala Asp Ala Val
Leu Ala Arg Val 130 135 140Tyr Lys Gln Ser Asp Leu Asp Thr Leu Ala
Lys Glu Ala Ser Ile Pro145 150 155 160Ile Ile Asn Gly Leu Ser Asp
Leu Tyr His Pro Ile Gln Ile Leu Ala 165 170 175Asp Tyr Leu Thr Leu
Gln Glu His Tyr Ser Ser Leu Lys Gly Leu Thr 180 185 190Leu Ser Trp
Ile Gly Asp Gly Asn Asn Ile Leu His Ser Ile Met Met 195 200 205Ser
Ala Ala Lys Phe Gly Met His Leu Gln Ala Ala Thr Pro Lys Gly 210 215
220Tyr Glu Pro Asp Ala Ser Val Thr Lys Leu Ala Glu Gln Tyr Ala
Lys225 230 235 240Glu Asn Gly Thr Lys Leu Leu Leu Thr Asn Asp Pro
Leu Glu Ala Ala 245 250 255His Gly Gly Asn Val Leu Ile Thr Asp Thr
Trp Ile Ser Met Gly Gln 260 265 270Glu Glu Glu Lys Lys Lys Arg Leu
Gln Ala Phe Gln Gly Tyr Gln Val 275 280 285Thr Met Lys Thr Ala Lys
Val Ala Ala Ser Asp Trp Thr Phe Leu His 290 295 300Cys Leu Pro Arg
Lys Pro Glu Glu Val Asp Asp Glu Val Phe Tyr Ser305 310 315 320Pro
Arg Ser Leu Val Phe Pro Glu Ala Glu Asn Arg Lys Trp Thr Ile 325 330
335Met Ala Val Met Val Ser Leu Leu Thr Asp Tyr Ser Pro Gln Leu Gln
340 345 350Lys Pro Lys Phe 35553364PRTArtificial
SequenceTAT-MTS(orf)-OTC delta linker 53Met Arg Lys Lys Arg Arg Gln
Arg Arg Arg Gly Ala Leu Val Ile Arg1 5 10 15Gly Ile Arg Asn Phe Asn
Leu Glu Asn Arg Ala Glu Arg Glu Ile Ser 20 25 30Lys Met Lys Pro Ser
Val Ala Pro Arg His Pro Ser Glu Phe Leu Lys 35 40 45Gly Arg Asp Leu
Leu Thr Leu Arg Asn Phe Thr Gly Glu Glu Ile Lys 50 55 60Tyr Met Leu
Trp Leu Ser Ala Asp Leu Lys Phe Arg Ile Lys Gln Lys65 70 75 80Gly
Glu Tyr Leu Pro Leu Leu Gln Gly Lys Ser Leu Gly Met Ile Phe 85 90
95Glu Lys Arg Ser Thr Arg Thr Arg Leu Ser Thr Glu Thr Gly Phe Ala
100 105 110Leu Leu Gly Gly His Pro Cys Phe Leu Thr Thr Gln Asp Ile
His Leu 115 120 125Gly Val Asn Glu Ser Leu Thr Asp Thr Ala Arg Val
Leu Ser Ser Met 130 135 140Ala Asp Ala Val Leu Ala Arg Val Tyr Lys
Gln Ser Asp Leu Asp Thr145 150 155 160Leu Ala Lys Glu Ala Ser Ile
Pro Ile Ile Asn Gly Leu Ser Asp Leu 165 170 175Tyr His Pro Ile Gln
Ile Leu Ala Asp Tyr Leu Thr Leu Gln Glu His 180 185 190Tyr Ser Ser
Leu Lys Gly Leu Thr Leu Ser Trp Ile Gly Asp Gly Asn 195 200 205Asn
Ile Leu His Ser Ile Met Met Ser Ala Ala Lys Phe Gly Met His 210 215
220Leu Gln Ala Ala Thr Pro Lys Gly Tyr Glu Pro Asp Ala Ser Val
Thr225 230 235 240Lys Leu Ala Glu Gln Tyr Ala Lys Glu Asn Gly Thr
Lys Leu Leu Leu 245 250 255Thr Asn Asp Pro Leu Glu Ala Ala His Gly
Gly Asn Val Leu Ile Thr 260 265 270Asp Thr Trp Ile Ser Met Gly Gln
Glu Glu Glu Lys Lys Lys Arg Leu 275 280 285Gln Ala Phe Gln Gly Tyr
Gln Val Thr Met Lys Thr Ala Lys Val Ala 290 295 300Ala Ser Asp Trp
Thr Phe Leu His Cys Leu Pro Arg Lys Pro Glu Glu305 310 315 320Val
Asp Asp Glu Val Phe Tyr Ser Pro Arg Ser Leu Val Phe Pro Glu 325 330
335Ala Glu Asn Arg Lys Trp Thr Ile Met Ala Val Met Val Ser Leu Leu
340 345 350Thr Asp Tyr Ser Pro Gln Leu Gln Lys Pro Lys Phe 355
36054365PRTArtificial SequenceTAT-MTS(lad)-OTC delta linker 54Met
Arg Lys Lys Arg Arg Gln Arg Arg Arg Gln Ser Trp Ser Arg Val1 5 10
15Tyr Cys Ser Leu Ala Lys Arg Gly His Phe Asn Arg Ile Ser His Gly
20 25 30Leu Gln Gly Leu Ser Ala Val Pro Leu Arg Thr Tyr Ala Glu Phe
Leu 35 40 45Lys Gly Arg Asp Leu Leu Thr Leu Arg Asn Phe Thr Gly Glu
Glu Ile 50 55 60Lys Tyr Met Leu Trp Leu Ser Ala Asp Leu Lys Phe Arg
Ile Lys Gln65 70 75 80Lys Gly Glu Tyr Leu Pro Leu Leu Gln Gly Lys
Ser Leu Gly Met Ile 85 90 95Phe Glu Lys Arg Ser Thr Arg Thr Arg Leu
Ser Thr Glu Thr Gly Phe 100 105 110Ala Leu Leu Gly Gly His Pro Cys
Phe Leu Thr Thr Gln Asp Ile His 115 120 125Leu Gly Val Asn Glu Ser
Leu Thr Asp Thr Ala Arg Val Leu Ser Ser 130 135 140Met Ala Asp Ala
Val Leu Ala Arg Val Tyr Lys Gln Ser Asp Leu Asp145 150 155 160Thr
Leu Ala Lys Glu Ala Ser Ile Pro Ile Ile Asn Gly Leu Ser Asp 165 170
175Leu Tyr His Pro Ile Gln Ile Leu Ala Asp Tyr Leu Thr Leu Gln Glu
180 185 190His Tyr Ser Ser Leu Lys Gly Leu Thr Leu Ser Trp Ile Gly
Asp Gly 195 200 205Asn Asn Ile Leu His Ser Ile Met Met Ser Ala Ala
Lys Phe Gly Met 210 215 220His Leu Gln Ala Ala Thr Pro Lys Gly Tyr
Glu Pro Asp Ala Ser Val225 230 235 240Thr Lys Leu Ala Glu Gln Tyr
Ala Lys Glu Asn Gly Thr Lys Leu Leu 245 250 255Leu Thr Asn Asp Pro
Leu Glu Ala Ala His Gly Gly Asn Val Leu Ile 260 265 270Thr Asp Thr
Trp Ile Ser Met Gly Gln Glu Glu Glu Lys Lys Lys Arg 275 280 285Leu
Gln Ala Phe Gln Gly Tyr Gln Val Thr Met Lys Thr Ala Lys Val 290 295
300Ala Ala Ser Asp Trp Thr Phe Leu His Cys Leu Pro Arg Lys Pro
Glu305 310 315 320Glu Val Asp Asp Glu Val Phe Tyr Ser Pro Arg Ser
Leu Val Phe Pro 325 330 335Glu Ala Glu Asn Arg Lys Trp Thr Ile Met
Ala Val Met Val Ser Leu 340 345 350Leu Thr Asp Tyr Ser Pro Gln Leu
Gln Lys Pro Lys Phe 355 360 365
* * * * *
References