U.S. patent application number 14/676846 was filed with the patent office on 2015-08-13 for importation of mitochodrial protein by an enhanced allotopic approach.
The applicant listed for this patent is INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE (INSERM). Invention is credited to Crystel BONNET, Marisol CORRAL-DEBRINSKI, Valerie KALTIMBACHER, Jose-Alain SAHEL.
Application Number | 20150225740 14/676846 |
Document ID | / |
Family ID | 37308339 |
Filed Date | 2015-08-13 |
United States Patent
Application |
20150225740 |
Kind Code |
A1 |
CORRAL-DEBRINSKI; Marisol ;
et al. |
August 13, 2015 |
IMPORTATION OF MITOCHODRIAL PROTEIN BY AN ENHANCED ALLOTOPIC
APPROACH
Abstract
An expression vector containing appropriate
mitochondrion-targeting sequences (MTS) and appropriate 3'UTR
sequences provides efficient and stable delivery of a mRNA encoding
a protein (CDS) to the mitochondrion of a mammalian cell. The MTS
and 3'UTR sequences guide the CDS mRNA from the nuclear compartment
of the cell to mitochondrion-bound polysomes, where the CDS is
translated. This provides an efficient translocation of a mature
functional protein into the mitochondria. A method of targeting
mRNA expressed in the nuclear compartment of a mammalian cell to
the mitochondrion is also provided. The vector and methods can be
used to treat defects in mitochondrial function.
Inventors: |
CORRAL-DEBRINSKI; Marisol;
(Montreuil, FR) ; SAHEL; Jose-Alain; (Paris,
FR) ; KALTIMBACHER; Valerie; (Fontenay Sous Bois,
FR) ; BONNET; Crystel; (Montrouge, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE
(INSERM) |
Paris Cedex 13 |
|
FR |
|
|
Family ID: |
37308339 |
Appl. No.: |
14/676846 |
Filed: |
April 2, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14323393 |
Jul 3, 2014 |
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14676846 |
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11913618 |
Jun 5, 2008 |
9017999 |
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PCT/EP2006/005323 |
May 3, 2006 |
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14323393 |
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60676933 |
May 3, 2005 |
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Current U.S.
Class: |
435/320.1 |
Current CPC
Class: |
A61K 48/005 20130101;
A61P 43/00 20180101; C12N 9/0089 20130101; C12N 9/1085 20130101;
A61K 48/0058 20130101; C12N 9/0053 20130101; A61K 48/0066 20130101;
C12N 15/85 20130101; C12N 2800/22 20130101; C12N 2810/80 20130101;
C12Y 109/03001 20130101; C07K 14/47 20130101; C12N 15/8509
20130101; C07K 2319/07 20130101 |
International
Class: |
C12N 15/85 20060101
C12N015/85 |
Claims
1. An expression vector configured for the efficient and stable
delivery of a protein into the mitochondrion of a mammalian cell,
the vector comprising: a mitochondrion-targeting nucleic acid
sequence (MTS); a nucleic acid sequence encoding said protein in
accordance with a universal genetic code (CDS); and a 3'UTR nucleic
acid sequence, located 3' of said CDS, wherein, said MTS comprises
a nucleic acid sequence encoding the peptide SEQ ID NO: 46, said
3'UTR nucleic acid sequence comprises the nucleic acid sequence SEQ
ID NO: 47, said vector does not comprise: any sequence identical to
a 3'UTR of a naturally occurring mRNA which is a
nuclear-transcribed but not mitochondrion-targeted mRNA, a cDNA
sequence of said 3'UTR of a naturally occurring mRNA, or a DNA
sequence coding for said 3'UTR of a naturally-occurring mRNA in
accordance with the universal genetic code, and said vector does
not utilize a post-translation importation pathway, but utilizes a
co-translation importation pathway from the nucleus to the
mitochondrion.
2. The expression vector of claim 1, wherein the CDS nucleic acid
sequence encodes naturally-occurring functional mitochondrial
protein ND4.
3. The expression vector of claim 1, wherein the nucleic acid
sequence encoding the peptide SEQ ID NO: 46 is SEQ ID NO: 30.
4. The expression vector of claim 2, wherein the CDS nucleic acid
sequence encoding ND4 is SEQ ID NO: 29.
5. The expression vector of claim 1, wherein said mammalian cell is
a human cell.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the field of cell biology,
molecular genetics, and medicine. It more particularly relates to
the importation of proteins into the mitochondrion of animal and
human cells.
BACKGROUND OF THE INVENTION
[0002] Mitochondria occupy a central position in the overall
metabolism of eukaryotic cells; hence the oxidative phosphorylation
(OXPHOS), the Krebs's cycle, the urea cycle, the heme biosynthesis
and the fatty acid oxidation take place within the organelle.
Recently, another major role for mitochondria in determining the
cellular life span was established, as they are recognized to be a
major early mediator in the apoptotic cascade. Mitochondria are
also a major producer of reactive oxygen species (ROS) causing
oxidative stress and therefore inducers of cell death.
[0003] Primary defects in mitochondrial function are implicated in
over 120 diseases and the list continues to grow, they encompass an
extraordinary assemblage of clinical problems, commonly involving
tissues that have high energy requirements, such as retina, heart,
muscle, kidney, pancreas and liver. Their incidence is estimated of
1 in 5,000 live births. Indeed, combining epidemiological data on
childhood and adult mitochondrial diseases suggests this prevalence
as minimum, and could be much higher. Therefore, mitochondrial
pathologies are considered among the most common genetically
determined diseases, and are a major health issue since they remain
inaccessible to both curative and palliative therapies.
[0004] Mitochondrion is assembled with proteins encoded by genes
distributed between mitochondrial and nuclear genomes. These genes
include those encoding the structural proteins of the respiratory
chain complexes I-V, their associated substrates and products, the
proteins necessary for mitochondrial biogenesis, the apparatus to
import cytoplasmically synthesized precursors and the proteins
necessary for mitochondrial assembly and turnover. Studies leading
to the identification of genes involved in mitochondrial disorders
have made considerable progress in the last decade. Indeed,
numerous mutations in both mitochondrial DNA and a number of
nuclear genes have been reported in association with a striking
diversity of clinical presentations.
[0005] Approximately half of human mitochondrial disorders are
caused by pathogenic point mutations of mtDNA, one-third of which
are located in coding genes. There is currently no treatment for
any of these disorders, a possible therapeutic approach is to
introduce in the nucleus a wild-type copy of the gene mutated in
the mitochondrial genome and import normal copies of the gene
product into mitochondria from the cytosol. This approach has been
termed "allotopic expression".
[0006] There have already some reports describing that engineered
nucleus-localized version of some mtDNA genes could be expressed in
mammalian cells. For example, in a Leigh's disease case, a plasmid
was constructed in which the mitochondrial targeting signal of the
nuclear encoded COX8 gene was appended to a recoded mitochondrial
ATP6 gene, mutated in patients. Stably transfected cells from
patients present an improvement of growth in galactose medium and a
mild increase in ATP synthesis, however the amount of Atp6 protein
imported into mitochondria was relatively low (18.5%), implying
that the precursor was not imported efficiently (Manfredi, G., et
al., Rescue of a deficiency in ATP synthesis by transfer of MTATP6,
a mitochondrial DNA-encoded gene to the nucleus. Nature Genet.,
2002. 30: p. 394-399).
[0007] Oco-Cassio and co-workers have demonstrated that allotopic
expression of apocytochrome b and ND4 into Cos-7 and HeLa cells,
did not lead to an efficient mitochondrial import of these proteins
(Oca-Cossio, J., et al., Limitations of allotopic expression of
mitochondrial genes in mammalian cells. Genetics, 2003. 165: p.
707-720).
[0008] Hence, up today important limitations are found to the
allotopic expression as a therapeutic approach and require
optimization to overcome the significant hurdles before it can be
applied in genetic therapy.
[0009] One hypothesis that can explain the poor import ability of
the mitochondrial protein is its high hydrophobicity. Thus, the
precursor synthesized in the cytoplasm remains stuck on the outer
mitochondrial membrane.
[0010] Mitochondria assembly depends on balanced synthesis of 13
proteins encoded by mtDNA with more than a thousand others encoded
by nuclear DNA. As the vast majority of mitochondrial polypeptides
are synthesized in the cytoplasm, there is the requirement for an
efficient and specific protein targeting system. This process
involves the transport of mRNAs from the nucleus to the surface of
mitochondria.
[0011] The inventors examined the possibility that allotopic
expression of DNA such as mtDNA could be optimized by a targeted
localization of the mRNA to the mitochondrial surface.
SUMMARY OF THE INVENTION
[0012] Mitochondrial proteins are encoded by nucleic acids which
are located in the mitochondrion, i.e. mitochondrial nucleic acids
(mtDNA, mtRNA), as well as by nucleic acids which originate from
the nucleus, i.e. nuclear nucleic acids (nDNA, nRNA).
[0013] The inventors describe an enhanced allotopic approach for
importation of proteins into the mitochondrion. The present
invention provides means, including compositions and methods, which
enable mitochondrial importation at enhanced efficiency and
stability compared to prior art techniques. The means of the
invention enable a targeted localization of the mRNA to the
mitochondrial surface.
[0014] Compared to prior art techniques, the means of the invention
enable the efficient and stable importation of protein into the
mitochondrion of an animal of human in need thereof, such as an
animal or human having a cellular dysfunction caused by one or
several mutations in a gene encoding a mitochondrial protein.
[0015] The inventors demonstrate that mRNA sorting to the
mitochondrial surface is an efficient way to proceed to such an
allotopic expression, and that this mRNA sorting can be controlled
by selecting appropriate mitochondrion-targeting sequence (MTS) and
appropriate 3'UTR sequences. The CDS sequence which codes for the
protein to be delivered into the mitochondrion is guided by these
appropriate MTS and 3'UTR sequences from the nuclear compartment to
the mitochondrion-bound polysomes (where the CDS is translated),
and aids in an efficient translocation of a mature functional
protein into the mitochondria.
[0016] The inventors demonstrate that, to obtain a stable
therapeutically-effective importation, both an appropriate MTS and
an appropriate 3'UTR should preferably be used.
[0017] Appropriate MTS and 3'UTR sequences correspond to those of
nuclearly-transcribed mitochondrially-targeted mRNAs. If a vector
is used, it is preferred that it does not contain any 3'UTR which
would correspond to the 3'UTR of a nuclearly-transcribed but
not-mitochondrially-targeted mRNAs. To the best of the inventors'
knowledge, all commercially-available vectors contain such a
not-mitochondrially-targeted mRNA; it is then preferred to delete
this inappropriate 3'UTR from the vector before use as
mitochondrial importer.
[0018] The means of the invention are especially adapted to animal
and human cells, and more particularly to mammalian cells. They
give access to therapeutically-effective means for such cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIGS. 1A and 1B: Map and sequence of COX10 MTS-nATP6,
SOD2MTS-nATP6, COX10 MTS-nATP6-COX10 3'UTR, SOD2MTS-nATP6-SOD2
3'UTR obtained in the pCMV-Tag 4A vector.
[0020] FIG. 1A. The four constructs are schematically
represented.
[0021] FIG. 1B. The COX10 MTS-nATP6 and SOD2MTS-nATP6 are
introduced at the EcoRI restriction site of the pCMV-tag4A vector.
EcoRI restriction sites are framed. The ATG of SOD2 MTS, COX10 MTS
and nATP6, are bold and underlined. The COX10 3'UTR and the SOD2
3'UTR are inserted at the PvuI and MluI restriction sites,
represented in bold. FLAG tag epitope is in italics.
[0022] FIG. 2: RT-PCR analyses of RNAs purified from HeLa
transfected cells
[0023] 100 ng of total RNAs were reversed transcribed and subjected
to 30 cycles of PCR amplification, 10% of amplified product were
subjected to agarose electrophoresis. [0024] 1: Transiently
transfected cells with the COX10 MTS-nATP6 vector (SV40 3'UTR).
[0025] 2: Transiently transfected cells with COX10 MTS-nATP6-COX10
3'UTR vector. [0026] In 3 and 4 we examined RNAs from the same
transfection experiment but in this case it represents the stably
transfected cells. [0027] 5: HeLa transfected cells with the empty
pCMV-tag4A vector. [0028] 6: Hela cells.
[0029] Specific oligonucleotide primers were used to detect hybrid
ATP6 mRNA in transfected cells, for the MTS COX10-ATP6 product MTS
COX10 and ATP6 ORF 3' were used (cf. Table 2 below, in example 1).
For the amplification of the complete ATP6 ORF and the entire COX10
3'UTR the ATP6 ORF 5' primer and the 3' UTR COX10 3' Primer were
used (cf. Table 2 below, in example 1). As internal control, the
steady-state levels of COX6c mRNA were examined in all the RNA
preparations using both COX6 primers shown in said Table 2.
[0030] FIG. 3: Subcellular localization of the recoded Atp6 protein
in HeLa cells
[0031] Stably transfected cells with either COX10 MTS-nATP6 (SV40
3'UTR) or COX10 MTS-nATP6-COX10 3'UTR vector were visualized by
indirect immunofluorescence using antibodies to Flag and ATP
synthase subunit beta. The punctuate pattern of Flag antibody
staining indicates that the fusion Atp6 protein is efficiently
transported to mitochondria in vivo, since the same pattern of
mitochondria labeling was observed with the beta subunit of ATP
synthase.
[0032] FIGS. 4A and 4B: nATP6 proteins are efficiently imported
into mitochondria in vivo.
[0033] FIG. 4A: Proteins were extracted from HeLa cells and HeLa
transfected cells (COX10 MTS-nATP6-SV40 3'UTR or COX10
MTS-nATP6-COX10 3'UTR vectors) and assayed for import in the
absence and presence of proteinase K (PK). Proteins were treated
with 200 .mu.g/ml of proteinase K at 0.degree. C. for 30 min. Then,
they were separated on 4-12% polyacrylamide SDS gel and transferred
into a nitrocellulose membrane. The resulting blot was probed with
mouse monoclonal anti-ATP synthase subunit alpha or mouse
monoclonal anti-Flag M2 antibodies.
[0034] FIG. 4B: Histograms of the amount of COX10 MTS-nATP6-Flag
and Atp synthase subunit alpha with or without proteinase K.
Signals from immunoblots were scanned and quantified by the
MultiAnalyst System (Bio-Rad). The amount of the mature ATP6
protein insensitive to proteinase K proteolysis is approximately
185% higher in cells transfected with COX10 MTS-nATP6-COX10 3'UTR
vector compared to cells expressing the COX10 MTS-nATP6 without
COX10 3'UTR but with the SV40 Poly A signal. Besides, the amount of
the mature form of the recoded ATP6 protein inside mitochondria is
very similar to the one measured for the naturally imported ATP
synthase subunit alpha, confirming that recoded ATP6 proteins are
efficiently translocated into the organelle.
[0035] FIGS. 5A and 5B: Map and sequence of COX10 MTS-nND1,
COX10MTS-nND4, COX10 MTS-nND1-COX10 3'UTR, COX10MTS-nND4-COX10
3'UTR obtained in the pCMV-Tag 4A vector.
[0036] FIG. 5A. The four constructs are schematically
represented.
[0037] FIG. 5B. The COX10 MTS-nND1 and COX10MTS-nND4 are introduced
at the XhoI/SalI restriction sites of the pCMV-tag4A vector. XhoI
and SalI restriction sites are framed. The ATG of COX10 MTS, nND1
and nND4, are bold and underlined. The COX10 3'UTR is inserted at
the PvuI and MluI restriction sites, represented in bold. FLAG tag
is in italics.
[0038] FIG. 6: Immunocytochemistry of G3460A LHON fibroblats
[0039] The fusion protein was visualized by indirect
immunofluorescence using antibodies to Flag. Indicative of
mitochondrial import, cells transfected with either COX10
MTS-nND1-SV40 3'UTR or COX10 MTS-nND1-COX10 3'UTR vectors exhibited
a typically punctuate staining pattern, also observed with the beta
subunit of ATP synthase, which localize in vivo to the inner
mitochondrial membrane. In contrast, cells transfected with the
empty pCMV-Tag 4A vector exhibited a very low intensity and diffuse
cytoplasmic staining when antibodies to Flag were used.
[0040] FIG. 7: Immunocytochemistry of G11778A LHON fibroblats
[0041] The fusion protein was visualized by indirect
immunofluorescence using antibodies to Flag. Cells transfected with
either COX10 MTS-nND4-SV40 3'UTR or COX10 MTS-nND4-COX10 3'UTR
vectors exhibited a typically punctate staining pattern, also
observed with the beta subunit of ATP synthase, which localize in
vivo to the inner mitochondrial membrane. This data indicates that
ND4 is efficiently imported into mitochondria. In contrast, cells
transfected with the empty pCMV-Tag 4A vector exhibited a very low
intensity and diffuse cytoplasmic staining when antibodies to Flag
were used.
[0042] FIG. 8: Growth in glucose-free medium of non-transfected
fibroblasts with the G3460A mutation and transfected fibroblasts
with the MTS COX10-nND1-COX10 3'UTR vector
[0043] Fibroblasts from LHON patients presenting the G3460A
mutation were stably transfected with the MTS COX10-nND1-COX10
3'UTR vector and examined for their ability to growth on DMEM
medium supplemented with 10 mM galactose. Non-transfected
fibroblasts (LHON G3460A ND1) show a severe growth defect on
galactose medium, the ability to grow on galactose was
significantly improved when the recoded nND1 protein is expressed
in stably transfected cells (LHON G3460A ND1+MTSCOX10-nND1). Cells
were photographed after 6 day culture.
[0044] FIG. 9: recoded CDS of mtDNA (SEQ ID NO: 27-29)
[0045] FIG. 9 shows the human nucleic acid coding sequence of the
mitochondrial ATP6, ND1, ND4, recoded according to the universal
genetic code (nATP6, nND1, nND4 of SEQ ID NO:27, 28 and 29,
respectively). The recoded ND1 and ND4 which are shown in FIG. 9
also take into account the preferred human codon usage (see example
2 below).
[0046] FIG. 10: illustrative human co-translational MTS and 3'UTR
(SEQ ID NOS: 30, 47, 31, 60)
[0047] FIG. 10 shows the sequence of human COX10 MTS (SEQ ID NO:
30), human COX10 3'UTR (SEQ ID NO: 47), human SOD2 MTS (SEQ ID NO:
31), and human SOD2 3'NTR (SEQ ID NO: 60).
TABLE-US-00001 TABLE 5 Nucleic acid MTS Nucleic acid 3'UTR COX10
SEQ ID NO: 30 SEQ ID NO: 47 SOD2 SEQ ID NO: 31 SEQ ID NO: 60
[0048] FIGS. 11A-11I show the protein sequence coded by
illustrative human mitochondrially-targeted mRNA, as well as their
respective MTS peptide sequences and their respective 3'UTR
sequences. ATCC accession number is indicated for each of these
protein sequences.
ACO2=Aconitase;
[0049] SOD2=Mitochondrial Superoxide dismutase; ATP5b=P synthase
subunit beta; UQCRFS1=Ubiquinol-cytochrome c reductase, Rieske
iron-sulfur polypeptide 1; NDUFV1=NADH-ubiquinone oxidoreductase 51
kDa subunit, mitochondrial precursor (Complex I-51KD)
(CI-51KD)(NADH dehydrogenase flavoprotein 1); NDUFV2=NADH
dehydrogenase (ubiquinone) flavoprotein 2, 24 kDa;
ALDH2=Mitochondrial aldehyde dehydrogenase 2 precursor; COX10=Heme
A:farnesyltransferase;
AK2=Adenylate Kinase 2.
[0050] SEQ ID NO are as follows:
TABLE-US-00002 TABLE 6 MTS peptide 3'UTR Protein ACO2 SEQ ID NO: 32
SEQ ID NO: 33 SEQ ID NO: 48 SOD2 SEQ ID NO: 34 SEQ ID NO: 35 SEQ ID
NO: 49 ATP5b SEQ ID NO: 36 SEQ ID NO: 37 SEQ ID NO: 50 UQCRFS1 SEQ
ID NO: 38 SEQ ID NO: 39 SEQ ID NO: 51 NDUFV1 SEQ ID NO: 40 SEQ ID
NO: 41 SEQ ID NO: 52 NDUFV2 SEQ ID NO: 42 SEQ ID NO: 43 SEQ ID NO:
53 ALDH2 SEQ ID NO: 44 SEQ ID NO: 45 SEQ ID NO: 54 COX10 SEQ ID NO:
46 SEQ ID NO: 59 SEQ ID NO: 55 AK2 SEQ ID NO: 57 SEQ ID NO: 56
[0051] FIGS. 12A, 12B, 12C: Subcellular distribution of hybrid ATP6
mRNAs in HeLa cells
[0052] A. Total RNAs extracted from cells expressing the
SOD2.sup.MTS ATP6-3'UTR.sup.SV40 (S.T 1 and S.T 2) or the
SOD2.sup.MTS ATP6-3'UTR.sup.SOD2 (S.T 3 and S.T 4) vectors were
subjected to RT-PCR analysis to reveal amounts of hybrid ATP6
(SOD2.sup.MTS ATP6) mRNAs and endogenous SOD2, ATP6 and COX6c
mRNAs. The amount of RNAs used for the reverse transcription, PCR
conditions and specific oligonucleotides used for each gene are
summarized in Table 9.
[0053] B. RNAs were purified from mitochondrion-bound polysomes
(M-P) and free-cytoplasmic polysomes (F-P) of stably transfected
cell lines with either SOD2.sup.MTS ATP6-3'UTR.sup.SV40 (S.T 1 and
S.T 2) or SOD2.sup.MTS ATP6-3'UTR.sup.SOD2 (S.T 3 and S.T 4)
vectors and subjected to RT-PCR analysis. The abundance of
endogenous ATP6, SOD2 and COX6c mRNAs was determined in each
polysomal population using the conditions shown in Table 9.
[0054] C. Densitometric analyses were performed using the Quantity
One Biorad software system.
[0055] The difference between the amounts of hybrid ATP6 mRNAs in
cells expressing respectively SOD2.sup.MTS ATP6-3'UTR.sup.SV40 or
SOD2.sup.MTS ATP6-3'UTR.sup.SOD2 constructions was significant
according to the paired Student's t-test (F<0.0034, n=6).
[0056] FIG. 13: Subcellular localization of the recoded Atp6
protein in vivo
[0057] Stably transfected cells with either the empty pCMV-tag4A
vector, SOD2.sup.MTS ATP6-3'UTR.sup.SV40 or SOD2.sup.MTS
ATP6-3'UTR.sup.SOD2 plasmids were visualized by indirect
immunofluorescence using antibodies to Flag and ATP synthase
subunit .alpha.. For each cell type visualized, a merged image in
association with DAPI staining is shown at the right panel.
Indicative of the mitochondrial localization of recoded ATP6
proteins, cells transfected with either SOD2.sup.MTS
ATP6-3'UTR.sup.SV40 or SOD2.sup.MTS ATP6-3'UTR.sup.SOD2 plasmids
showed a significant colocalization of both Flag and ATP synthase
.alpha. signals. In contrast, cells transfected with the empty
vector exhibited a low diffuse cytoplasmic staining.
[0058] FIGS. 14A, 14B, 14C: Recoded ATP6 proteins are efficiently
imported into mitochondria in vivo.
[0059] A. Six independent mitochondria purifications were performed
with cells stably transfected with either SOD2.sup.MTS
ATP6-3'UTR.sup.SV40 or SOD2.sup.MTS ATP6-3'UTR.sup.SOD2 plasmids
and subjected to Western blot analysis. Signals for the ATP6
precursors and mature forms were scanned and quantified by the
Quantity One System (Bio-Rad). No significant differences between
the amounts of the precursor and the mature form of the recoded
ATP6 proteins were observed in each cell line examined.
[0060] B. Upper panel: Schematic representation of mitochondrial
import intermediates. The hydrophobic passenger protein can be
trapped en route to the matrix. In this step, the protein can be
blocked or represented an intermediate of translocation. This
doesn't prevent the cleavage of the MTS by a mitochondrial
processing peptidase, the rest of the protein remains accessible to
PK digestion and therefore if digested it becomes undetectable in
the Western blot assay. The fraction of the protein completely
translocated turns into a mature protein insensitive to PK located
in the inner mitochondrial membrane. MM: mitochondrial matrix; OM:
outer membrane; MIS: mitochondrial intermembrane space, TOM:
Translocase of the Outer Membrane, TIM: Translocase of the Inner
membrane.
[0061] Middle panel: Mitochondria extracted from transfected cells
with either the empty pCMV-Tag 4A vector, SOD2.sup.MTS
ATP6-3'UTR.sup.SV40 or SOD2.sup.MTS ATP6-3'UTR.sup.SOD2 plasmids
were subjected to Western blot essays. 20 .mu.g of proteins were
treated with 150 .mu.g/ml of PK at 0.degree. C. for 30 minutes and
subjected to immunoblotting analysis using anti-ATP synthase
subunit .alpha. and anti-Flag M2 antibodies. Densitometric analyses
of experiments performed with six independent mitochondrial
purifications were represented at the lower panel. We normalized
values measured for the signal of the mature form of ATP6 resistant
to PK with ATP.alpha. signal revealed after PK digestion. We then
compared the value obtained for cells expressing either the
SOD2.sup.MTS ATP6-3'UTR.sup.SV40 or the SOD2.sup.MTS
ATP6-3'UTR.sup.SOD2 plasmids. Signals from Western blots were
scanned and quantified by the Quantity One System (Bio-Rad). The
difference between the amounts of fully mitochondrial translocated
ATP6 protein in cells expressing respectively SOD2.sup.MTS
ATP6-3'UTR.sup.SV40 or SOD2.sup.MTS ATP6-3'UTR.sup.SOD2
constructions was significant according to the paired Student's
t-test (P<0.0022, n=6).
[0062] C. 20 .mu.g of mitochondria isolated from cells stably
transfected with SOD2.sup.MTS ATP6-3'UTR.sup.SOD2 vector treated
with 150 .mu.g/ml of PK and 1% Triton X100 at 0.degree. C. for 30
min and subsequently subjected to Western analysis.
[0063] FIG. 15: Mitochondrial import ability of ATP6 proteins based
on the mesohydrophobicity index
[0064] A plot developed by Claros and Vincens was used to measure
mitochondrial import ability of fusion ATP6 proteins. By this
approach, the fusion SOD2.sup.MTSATP6 protein would not be
importable. Mesohydrophobicity, which is the average regional
hydrophobicity over a 69 amino acid region, was calculated using
Mito-ProtII. Values obtained are the following: ATP6: 1.41;
SOD2.sup.MTSATP6: 1.41; COX8.sup.MTSATP6: 1.41; SOD2: -1.26; COX8:
-1.63.
[0065] FIG. 16: rescue of NARP cells; survival rate on galactose
medium of NARP cells (mutated ATP6), and of NARP cells transfected
by a SOD2 MTS-ATP vector (SOD2 MTS-ATP6-SV40 3'UTR), or by a vector
of the invention (SOD2 MTS-ATP6-SOD2 3'UTR); see also example 3,
table 11.
[0066] FIG. 17: rescue of LHON fibroblasts; survival rate on
galactose medium of LHON fibroblasts (mutated ND1), and of LHON
fibroblasts transfected by a COX10 MTS-ND1-SV40 3'UTR vector, or by
a vector of the invention (COX10 MTS-ND1-COX10 3'UTR); see also
example 4, table 12.
[0067] FIG. 18: mitochondrial distribution in retinal gangion cells
(RGC) transfected with the mutated version of ND1.
[0068] FIG. 19: rescue of NARP cells; anti-Flag, Mito-tracker and
merged+DAPI staining of NARP cells transfected either with SOD2
MTS-ATP6-SV40 3' UTR, or with SOD2 MTS-ATP6-SOD2 3'UTR.
DETAILED DESCRIPTION OF THE INVENTION
[0069] The present invention relates to the use and control of mRNA
sorting at the surface of mitochondria.
[0070] Schematically, the present invention relates to the use of
nucleic acid sequences corresponding to a co-translational MTS and
of a co-translational 3'UTR, for guiding a desired mRNA (which
codes a desired mitochondrial protein) from the nucleus to the
mitochondria-bound polysomes, and for inducing the effective
translocation of the translated protein into the mitochondrion.
[0071] By "co-translational", it is herein referred to a
nuclearly-encoded mitochondrially-targeted pathway.
Mitochondrion-Targeting Sequences (MTS):
[0072] Sequences known as mitochondrion-targeting signal or
mitochondrial targeting signal are referred to as MTS by the
skilled person.
[0073] A MTS sequence can be identified within a protein or nucleic
acid sequence by a person of ordinary skill in the art.
[0074] Most mitochondrion-targeting peptides consist of a
N-terminal pre-sequence of about 15 to 100 residues, preferably of
about 20 to 80 residues. They are enriched in arginine, leucine,
serine and alanine. Mitochondrial pre-sequences show a statistical
bias of positively charged amino acid residues, provided mostly
through arginine residues; very few sequences contain negatively
charged amino acids. Mitochondrion-targeting peptides also share an
ability to form an amphilic alpha-helix.
[0075] A complete description of a method to identify a MTS is
available in: M. G. Claros, P. Vincens, 1996 (Eur. J. Biochem. 241,
779-786 (1996), "Computational method to predict mitochondrially
imported proteins and their targeting sequences"), the content of
which is herein incorporated by reference.
[0076] Software is available to the skilled person to identify the
MTS of a given sequence. Illustrative software notably comprises
the MitoProt.RTM. software, which is available e.g. on the web site
of the Institut fur Humangenetik; Technische Universitat Munchen,
Germany. The MitoProt.RTM. software calculates the N-terminal
protein region that can support a Mitochondrial Targeting Sequence
and the cleavage site. The identification of the N-terminal
mitochondrial targeting peptide that is present within a protein
gives a direct access to the nucleic acid sequence, i.e. to the MTS
(e.g. by reading the corresponding positions in the nucleic acid
sequence coding for said protein).
[0077] Illustrative human MTS peptide sequences and human 3'UTR
originating from human nuclearly-encoded mitochondrially-targeted
mRNA are given in FIGS. 11A, 11B, 11C, 11D, 11E, 11F, 11G, 11H and
11I.
[0078] SEQ ID NOs are as follows:
TABLE-US-00003 TABLE 7 Illustrative human mRNAs which are
nuclearly-encoded but mitochondrially-targeted MTS peptide Figure
ACO2 SEQ ID NO: 32 11A SOD2 SEQ ID NO: 34 11B ATP5b SEQ ID NO: 36
11C UQCRFS1 SEQ ID NO: 48 11D NDUFV1 SEQ ID NO: 40 11E NDUFV2 SEQ
ID NO: 42 11F ALDH2 SEQ ID NO: 44 11G COX10 SEQ ID NO: 46 11H
3'UTR:
[0079] The 3'UTR of a RNA molecule is defined as the fragment of
this RNA molecule that extends from the STOP codon to the end of
the molecule. According to the universal genetic code, there are
three possible STOP codons: TGA, TAA, TAG.
[0080] An online data base gives direct access to 3'UTR
sequences.
[0081] Illustrative 3'UTR sequences which can used in accordance
with the invention are shown in FIGS. 11A, 11B, 11C, 11D, 11E, 11F,
11G, 11H and 11I (Accession numbers of these sequences are also
indicated).
[0082] SEQ ID NOs are as follows:
TABLE-US-00004 TABLE 8 Illustrative human mRNAs which are
nuclearly-encoded but mitochondrially-targeted 3'UTR Figure ACO2
SEQ ID NO: 33 11A SOD2 SEQ ID NO: 35 11B ATP5b SEQ ID NO: 37 11C
UQCRFS1 SEQ ID NO: 39 11D NDUFV1 SEQ ID NO: 41 11E NDUFV2 SEQ ID
NO: 43 11F ALDH2 SEQ ID NO: 45 11G COX10 SEQ ID NO: 47 10 AK2 SEQ
ID NO: 57 11I
Vectors of the Invention:
[0083] The present invention relates to a vector which is adapted
to the efficient and stable delivery of a protein into the
mitochondrion of an animal or human cell, preferably a mammalian
cell, most preferably a human cell.
[0084] The vector of the invention can be produced in the form of a
recombinant vector. Advantageously, the vector of the invention is
an expression vector.
[0085] A vector of the invention comprises: [0086] at least one
nucleic acid sequence encoding a mitochondrion-targeting signal
(also referred to as: MTS nucleic acid sequence), [0087] at least
one nucleic acid sequence which encodes said protein to be
delivered, in accordance with the universal genetic code (also
referred to as: CDS), and [0088] at least one 3' nucleic acid
sequence.
[0089] Said at least one MTS nucleic acid sequence is a
co-translational MTS nucleic acid sequence, or a conservative
fragment or variant thereof.
[0090] Said at least one 3' nucleic acid sequence is a
co-translational 3'UTR nucleic acid sequence or the DNA sequence of
such a co-translational 3'UTR, or a conservative fragment or
variant thereof.
[0091] Preferably, said vector does not comprise a
non-co-translational 3'UTR.
[0092] Said vector does not use a post-translation importation
pathway, but uses a co-translation importation pathway from nucleus
to said mitochondrion.
[0093] The delivery of protein according to the invention not only
comprises the translocation of the protein-encoding nucleic acid
from nucleus towards mitochondrion, but also comprises the
translation of the encoded protein in the cytosol but at proximity
of the mitochondrion (on mitochondrion-bound polysomes), and the
effective importation of the translated protein into said
mitochondrion. The invention provides a very advantageous
importation mechanism compared to prior art techniques, which
provided the mitochondrion with mature proteins at an
unsatisfactory level of efficiency.
[0094] The present invention further provides a stable importation
of said protein into the mitochondrion. It means that the protein
rescue obtained by the invention is a rescue that is stable over
time: the fibroblasts of a LHON patient transfected by a vector of
the invention (expressing ND1) has grown in vitro for at least 20
days on a galactose culture medium. To the best of the inventors'
knowledge, this is the first time that a culture of LHON patient
fibroblasts can be kept growing for such a long period on a
galactose medium.
[0095] The invention thus relates to a vector adapted to the
efficient and stable delivery of a protein into the mitochondrion
of an animal or human cell, preferably a mammalian cell, most
preferably a human cell, which comprises: [0096] at least one
mitochondrion-targeting nucleic acid sequence (MTS nucleic acid
sequence), [0097] at least one nucleic acid sequence which encodes
said protein in accordance with the universal genetic code (CDS),
and [0098] at least one 3' nucleic acid sequence, which is located
in 3' of said at least one MTS nucleic acid sequence and said at
least one CDS.
[0099] Preferably, said at least MTS nucleic acid sequence is in 5'
position compared to said at least one CDS sequence, whereby the
vector has the following structure (from 5' to 3'): at least one
MTS nucleic acid sequence-at least one CDS-at least one 3'UTR.
[0100] Said at least one MTS nucleic acid sequence is: [0101] the
MTS RNA sequence of a nuclearly-encoded mitochondrially-targeted
mRNA, preferably the MTS RNA sequence of a naturally-occurring
nuclearly-encoded mitochondrially-targeted mRNA, or [0102] the cDNA
sequence of such a MTS RNA sequence, or [0103] a DNA sequence
coding for such a MTS RNA sequence in accordance with the universal
genetic code, or [0104] a conservative variant or fragment of such
a RNA or cDNA or DNA MTS sequence, which derives therefrom by
deletion and/or substitution and/or addition of one or several
nucleotides, but has retained a mitochondrion-targeting
function.
[0105] In other words, said at least one MTS nucleic acid sequence
is: [0106] the RNA sequence of a MTS which targets a (preferably
naturally-occurring) nuclearly-transcribed mRNA to the surface of a
mitochondrion in a cell collected from a healthy animal or human
being, or in a normal animal or human cell, or [0107] the cDNA
sequence of such a MTS RNA sequence, or [0108] a DNA sequence
coding for such a MTS RNA sequence in accordance with the universal
genetic code, or [0109] a conservative variant or fragment of such
a RNA or cDNA or DNA MTS sequence, which derives therefrom by
deletion and/or substitution and/or addition of one or several
nucleotides, but has retained a mitochondrion-targeting
function.
[0110] Preferably, said at least one MTS nucleic acid sequence is:
[0111] the cDNA sequence of a MTS of a nuclearly-encoded
mitochondrially-targeted m RNA, or [0112] a conservative variant or
fragment of such a cDNA sequence, which derives therefrom by
deletion and/or substitution and/or addition of one or several
nucleotides, but has retained a mitochondrion-targeting
function.
[0113] Said at least one 3' nucleic acid sequence is: [0114] the
3'UTR sequence of a nuclearly-encoded mitochondrially-targeted
mRNA, preferably the 3'UTR sequence of a naturally-occurring
nuclearly-encoded mitochondrially-targeted mRNA, or [0115] the cDNA
sequence of such a 3'UTR sequence, or [0116] a DNA sequence coding
for such a 3'UTR sequence in accordance with the universal genetic
code, or [0117] a conservative variant or fragment of such a RNA or
cDNA or DNA 3'UTR sequence, which derives therefrom by deletion
and/or substitution and/or addition of one or several nucleotides,
and which, when replacing the wild-type 3'UTR of said
nuclearly-encoded mitochondrially-targeted mRNA, still allows for a
mitochondrial targeting of the resulting mRNA.
[0118] In other words, said at least one 3' nucleic acid sequence
is: [0119] the RNA sequence of the 3'UTR of a nuclearly-transcribed
mitochondrially-targeted mRNA, i.e. the RNA sequence of the 3'UTR
of a (preferably naturally-occurring) nuclearly-transcribed RNA
which is targeted to the surface of a mitochondrion in a cell
collected from a healthy animal or human being, or in a normal
animal or human cell, or [0120] the cDNA sequence of such a 3'UTR
sequence, or [0121] a DNA sequence coding for such a 3'UTR sequence
in accordance with the universal genetic code, or [0122] a
conservative variant or fragment of such a RNA or cDNA or DNA 3'UTR
sequence, which derives therefrom by deletion and/or substitution
and/or addition of one or several nucleotides, and which, when
replacing the wild-type 3'UTR of said nuclearly-encoded
mitochondrially-targeted mRNA, still allows for a mitochondrial
targeting of the resulting mRNA.
[0123] Preferably, said at least one 3' nucleic acid sequence is:
[0124] the cDNA sequence of the 3'UTR sequence of a
nuclearly-encoded mitochondrially-targeted mRNA, or [0125] a
conservative variant or fragment of such a cDNA sequence, which
derives therefrom by deletion and/or substitution and/or addition
of one or several nucleotides, and which, when replacing the
wild-type 3'UTR of said nuclearly-encoded mitochondrially-targeted
mRNA, still allows for a mitochondrial targeting of the resulting
mRNA.
[0126] The resulting vector does not use a post-translation
importation pathway, but uses a co-translation importation pathway
from nucleus to said mitochondrion.
[0127] Preferably, said vector (inserted nucleic acid construct
included) does not comprise any sequence which would be identical
to: [0128] the 3'UTR of a naturally-occurring mRNA which is a
(preferably naturally-occurring) nuclearly-transcribed but
non-mitochondrially-targeted mRNA, or [0129] the cDNA sequence of
such a 3'UTR sequence, or [0130] a DNA sequence coding such a 3'UTR
in accordance with the universal genetic code.
[0131] Preferably, said vector (inserted nucleic acid construct
included) does not comprise any sequence which would be identical
to: [0132] the 3'UTR of a mRNA which is not targeted to the surface
of a mitochondrion, and preferably the 3'UTR of a
naturally-occurring mRNA which is not targeted to the surface of a
mitochondrion, or [0133] the cDNA sequence of such a 3'UTR
sequence, or [0134] a DNA sequence coding such a
naturally-occurring mRNA 3'UTR in accordance with the universal
genetic code.
[0135] The present invention more particularly relates to a vector
adapted to the efficient and stable delivery of a protein into the
mitochondrion of a mammalian cell, which comprises: [0136] at least
one mitochondrion-targeting nucleic acid sequence (referred to as
MTS nucleic acid sequence), [0137] at least one nucleic acid
sequence which encodes said protein in accordance with the
universal genetic code (referred to as CDS sequence), and [0138] at
least one 3' nucleic acid sequence, which is located in 3' of said
at least one MTS nucleic acid sequence and of said at least one
CDS, wherein said at least one MTS nucleic acid sequence is: [0139]
the cDNA sequence of a MTS of a nuclearly-encoded
mitochondrially-targeted mRNA, or [0140] a conservative variant or
fragment of such a cDNA sequence, which derives therefrom by
deletion and/or substitution and/or addition of one or several
nucleotides, but has retained a mitochondrion-targeting function,
wherein said at least one 3' nucleic acid sequence is: [0141] the
cDNA sequence of the 3'UTR sequence of a nuclearly-encoded
mitochondrially-targeted mRNA, or [0142] a conservative variant or
fragment of such a cDNA sequence, which derives therefrom by
deletion and/or substitution and/or addition of one or several
nucleotides, and which, when replacing the wild-type 3'UTR of said
naturally-occurring mRNA, still allows for a mitochondrial
targeting of the resulting mRNA, wherein said vector does not
comprise any sequence which would be identical to: [0143] the 3'UTR
of a naturally-occurring mRNA which is a nuclearly-transcribed but
not-mitochondrially-targeted mRNA, or [0144] the cDNA sequence of
such a 3'UTR sequence, or [0145] a DNA sequence coding for such a
naturally-occurring mRNA 3'UTR in accordance with the universal
genetic code, whereby said vector does not use a post-translation
importation pathway, but uses a co-translation importation pathway
from nucleus to said mitochondrion.
[0146] Said at least one MTS nucleic acid sequence can e.g. be the
MTS nucleic acid sequence of ACO2, or of SOD2, or of ATP5b, or of
UQCRFS1, or of NDUFV1, or of NDUFV2, or of ALDH2, or of COX10.
[0147] Said at least one MTS nucleic acid sequence may thus code
for a sequence of SEQ ID NO:32, or SEQ ID NO:34, or SEQ ID NO:36,
or SEQ ID NO:38, or SEQ ID NO:40, or SEQ ID NO:42, or SEQ ID NO:44,
or SEQ ID NO:46 (=the MTS peptidic or polypeptidic sequence of
human ACO2, SOD2, ATP5b, UQCRFS1, NDUFV1, NDUFV2, ALDH2, COX10,
respectively; see FIGS. 11A-11H).
[0148] Preferably, said at least one MTS nucleic acid sequence is
the MTS nucleic acid sequence of ACO2, or of SOD2, or of ATP5b, or
of COX10.
[0149] Said at least one MTS nucleic acid sequence may thus code
for a sequence of SEQ ID NO:32, or SEQ ID NO:34, or SEQ ID NO:36,
or SEQ ID NO:46 (=the MTS peptidic or polypeptidic sequence of
human ACO2, SOD2, ATP5b, COX10, respectively).
[0150] Preferably, said at least one MTS nucleic acid sequence is
SEQ ID NO: 30, or SEQ ID NO: 31 (MTS nucleic acid sequence of human
COX10 and SOD2, respectively; see FIG. 10).
[0151] Said at least one 3' nucleic acid sequence can be e.g. be:
[0152] the 3'UTR sequence of ACO2, or of SOD2, or of ATP5b, or of
UQCRFS1, or of NDUFV1, or of NDUFV2, or of ALDH2, or of COX10, or
of AK2, or [0153] the cDNA sequence of such a 3'UTR sequence, or
[0154] a DNA sequence coding for such a 3'UTR sequence in
accordance with the universal genetic code.
[0155] Said at least one 3' nucleic acid sequence may thus comprise
or consist of those of SEQ ID NO: 33, or SEQ ID NO: 35, or SEQ ID
NO: 37, or SEQ ID NO: 39, or SEQ ID NO: 41, or SEQ ID NO: 43, or
SEQ ID NO: 45, or SEQ ID NO: 47, or SEQ ID NO: 57 (=the sequences
corresponding to the human 3'UTR of ACO2, SOD2, ATP5b, UQCRFS1,
NDUFV1, NDUFV2, ALDH2, COX10, AK2, respectively; see FIGS. 10 and
11A-11I).
[0156] Preferably, said at least one 3' nucleic acid sequence is:
[0157] the 3'UTR sequence of ACO2, or of SOD2, or of ATP5b, or of
COX10, or of AK2, or [0158] the cDNA sequence of such a 3'UTR, or
[0159] a DNA sequence coding for such a 3'UTR sequence.
[0160] Said at least one 3' nucleic acid sequence may thus comprise
or consist of SEQ ID NO: 33, or SEQ ID NO: 35, or SEQ ID NO: 37, or
SEQ ID NO: 47, or SEQ ID NO: 57 (=the sequences corresponding to
the human 3'UTR of ACO2, SOD2, ATP5b, COX10, AK2, respectively; see
FIGS. 10 and 11A-11I).
[0161] Preferably, said at least one 3' nucleic acid sequence is
SEQ ID NO: 35 (human SOD2 3'UTR), or SEQ ID NO: 47 (human COX10
3'UTR).
[0162] Said at least one CDS nucleic acid sequence can be a RNA, a
cDNA or a DNA sequence. Preferably, said at least one CDS sequence
is a cDNA sequence.
[0163] According to a very advantageous aspect of the invention,
said at least one CDS may be any nucleic acid which codes for a
protein that may be found useful for a mitochondrion. Contrary to
prior art techniques, the technology of the invention is indeed not
limited by the level of hydrophobicity of the encoded protein.
[0164] Said at least one CDS may thus be any nucleic acid coding
for a mitochondrial protein. This nucleic acid may be a
mitochondrial nucleic acid, or a nuclear nucleic acid coding for a
mitochondrial protein.
[0165] Most preferably said at least one CDS sequence codes for a
naturally-occurring functional mitochondrial protein, such as Cox1,
Cox2, Cox3, Atp6, Atp8, Cytb, Nd1, Nd2, Nd3, Nd4, Nd41, Nd5,
Nd6.
[0166] Preferably, said at least one CDS sequence is the sequence
of a naturally-occurring mitochondrial nucleic acid, recoded in
accordance with the universal genetic code.
[0167] The mitochondrial nucleic acids use a mitochondrial genetic
code which is slightly different from the universal genetic code
that is used by nuclear nucleic acids.
[0168] When the protein to be imported into said mitochondrion
corresponds to a naturally-occurring mitochondrial protein, the
naturally-occurring form of its nucleic acid sequence follows the
mitochondrial genetic code.
[0169] When such a mitochondrial nucleic acid has to be inserted in
the vector of the invention, the mitochondrial nucleic acid
sequence has to be recoded in accordance with the universal genetic
code, as the vector directs a co-translational importation process
from nucleus to mitochondrion. Hence, a nuclear-encoded version of
the mitochondrial nucleic acid sequence has to be created. This
nuclear-encoded version can be produced by codon substitution in
the mitochondrial nucleic acid, so as to replace those codons which
are read by the mitochondrial genetic system with codons of the
universal genetic code. For example, the mammalian UGA codon
directs insertion of a tryptophan in mitochondria, but is a stop
codon in the nuclear genetic code. Therefore, the UGA codon of a
mitochondrial nucleic acid has to be replaced with UGG which codes
for tryptophan in the universal genetic code.
TABLE-US-00005 TABLE 4 universal vs. mitochondrial genetic code
codon Universal code Human mitochondrial code UGA Stop Trp AGA Arg
Stop AGG Arg Stop AUA Ile Met
[0170] Codon usage in mitochondria vs. the universal genetic code
is described in Lewin, Genes V, Oxford University Press; New York
1994, the content of which being incorporated by reference.
[0171] Codon substitutions notably include: [0172] UGA to UGG,
[0173] AGA to UAA, UAG or UGA, [0174] AGG to UAA, UAG or UGA,
[0175] AUA to AUG, CUG or GUG, [0176] AUU to AUG, CUG or GUG.
[0177] Said at least one CDS sequence may e.g. a nucleic acid
sequence coding for Atp6, or Nd1, or Nd4, such as a nucleic acid
sequence of ATP6, or of ND1, or of ND4, recoded in accordance with
the universal genetic code (e.g. a sequence of SEQ ID NO:27, NO:28
or NO:29, see FIG. 9).
[0178] Said at least one CDS sequence may e.g. a nucleic acid
sequence a nucleic acid sequence coding for Cox1, Cox2, Cox3, Atp8,
Cytb, Nd2, Nd3, Nd41, Nd5, Nd6, such as a nucleic acid sequence of
COX1, COX2, COX3, ATP8, Cytb, ND2, ND3, ND41, ND5, ND6.
[0179] The description of the thirteen naturally-occurring
mitochondrial nucleic acids can be found in Andrew et al. 1999 (Nat
Genet. 1999 October; 23(2): 147).
[0180] Preferably, said recoding is made taking into account the
preferred usage codon of said mammalian cell, and most preferably
taking into account the human preferred usage codon.
[0181] When recoding mitochondrial nucleic acid according to the
universal genetic code, it is according to the present invention
very advantageous to take into account the preferred codon usage of
the subject or patient, to which the vector or nucleic acid of the
invention is to be administered.
[0182] Preferred codon usage principles, as well as examples of
preferred codon usage for various organisms can e.g. be found in
Klump and Maeder, 1991 (Pure & Appl. Chem., vol. 63, No. 10,
pp. 1357-1366 "the thermodynamic basis of the genetic code"), the
content of which is herein incorporated by reference. An
illustrative preferred codon usage for human beings is shown in
Table 3 below (see example 2).
[0183] Said at least one CDS sequence may e.g. the nucleic acid
sequence of SEQ ID NO:28 or of SEQ ID NO:29 (i.e. a nucleic acid
sequence of ND1 or of ND4, recoded in accordance with the universal
genetic code, and taking into account the human preferred usage
codon).
[0184] The vector of the invention may e.g. comprise: [0185] at
least one SOD2 MTS nucleic acid sequence and at least one SOD2
3'UTR, or [0186] at least one COX10 MTS nucleic acid sequence and
at least one COX10 3'UTR, or [0187] any combination of these MTS
nucleic acid sequences and 3'UTR that the skilled person may find
appropriate.
[0188] Such a vector may e.g. comprise a recoded ATP6, ND1 or ND4
as CDS.
[0189] The vector of the invention may e.g. comprise at least one
sequence of SEQ ID NO:21 (COX10 MTS-re-coded ATP6-COX10 3'UTR), SEQ
ID NO:22 (SOD2 MTS-re-coded ATP6-SOD2 3'UTR), SEQ ID NO:25 (COX10
MTS-re-coded ND1-COX10 3'UTR), SEQ ID NO:25 (COX10 MTS-re-coded
ND4-COX10 3'UTR).
[0190] Alternatively, said at least one CDS sequence may be the
nucleic acid sequence of a nuclear nucleic acid which encodes a
functional mitochondrial protein, e.g., a naturally-occurring
nuclear nucleic acid which encodes a functional mitochondrial
protein.
[0191] More particularly, said at least one nuclear nucleic acid
can be a nuclearly-transcribed mitochondrially-targeted mRNA, or
the cDNA sequence of such a mRNA, or the DNA sequence coding for
such a mRNA.
[0192] More particularly, said at least one nuclear nucleic acid
can be a nuclearly-transcribed mRNA which is not
mitochondrially-targeted, or the cDNA sequence of such a mRNA, or
the DNA sequence coding for such a mRNA.
[0193] Said vector may further comprise one or several expression
control sequences.
[0194] The selection of suitable expression control sequences, such
as promoters is well known in the art, as is the selection of
appropriate expression vectors (see e.g. Sambrook et al. "Molecular
Cloning: A laboratory Manual", 2.sup.nd ed., vols. 1-3, Cold Spring
Harbor Laboratory, 1989, the content of which is herein
incorporated by reference).
[0195] Said vector may thus further comprise at least one promoter
operably linked to said at least one MTS sequence, said at least
one CDS sequence, said at least one 3' sequence.
[0196] Said promoter may e.g. be a constitutive promoter, such as
e.g. a CMV promoter.
[0197] Said vector may further comprise a termination site.
[0198] Said vector may further comprise one of several of the
following expression control sequences: insulators, silencers,
IRES, enhancers, initiation sites, termination signals.
[0199] Said vector may further comprise an origin of
replication.
[0200] Preferably, said promoter and said origin of replication are
adapted to the transduction or infection of animal or human cells,
preferably to the transduction or infection of human cells.
[0201] Said vector can e.g. a plasmid, or a virus, such as an
integrating viral vector, e.g. a retrovirus, an adeno-associated
virus (AAV), or a lentivirus, or is a non-integrating viral vector,
such as an adenovirus, an alphavirus, a Herpes Simplex Virus
(HSV).
[0202] Said vector may further comprise a nucleic acid coding for a
detectable marker, such as a FLAG epitope or green fluorescent
protein (GFP)
[0203] The present invention also relates to a process for the
production of a vector of the invention, which comprises: [0204]
providing a vector, and depleting from its original 3'UTR, if any,
[0205] inserting in this vector at least one MTS nucleic acid
sequence, at least one CDS sequence, and at least one 3' sequence
as above-described.
[0206] As already-mentioned, said vector should preferably not
comprise any sequence corresponding to the 3'UTR of a
nuclearly-encoded but non-mitochondrially-targeted mRNA. To the
best of the inventors' knowledge, all commercially-available
vectors contain such an inappropriate 3'UTR; according to the
present invention, such a 3'UTR should hence be removed from the
vector. It may e.g. be replaced by an appropriate 3' sequence
corresponding to a nuclearly-encoded and mitochondrially-targeted
mRNA.
Nucleic Acid Construct of the Invention:
[0207] The nucleic acid construct which is carried by the vector of
the invention is also encompassed by the present invention. The
present invention more particularly relates to a non-naturally
occurring nucleic acid construct.
[0208] A non-naturally occurring nucleic acid construct of the
invention comprises: [0209] at least one mitochondrion-targeting
nucleic acid sequence (referred to as MTS nucleic acid sequence),
[0210] at least one nucleic acid sequence which encodes said
protein in accordance with the universal genetic code (referred to
as CDS sequence), and [0211] at least one 3' nucleic acid sequence,
which is located in 3' of said at least one MTS nucleic acid
sequence and of said at least one CDS sequence.
[0212] Said at least one MTS nucleic acid sequence is: [0213] the
MTS RNA sequence of a nuclearly-encoded mitochondrially-targeted
mRNA, such as the MTS RNA sequence of a naturally-occurring
nuclearly-encoded mitochondrially-targeted mRNA, or [0214] the cDNA
sequence of such a RNA, or [0215] a DNA sequence coding for such a
MTS RNA sequence, or [0216] a conservative variant or fragment of
such a RNA or DNA MTS sequence, which derives therefrom by deletion
and/or substitution and/or addition of one or several nucleotides,
but has retained a mitochondrion-targeting function.
[0217] Said at least one 3' nucleic acid sequence is: [0218] the
3'UTR sequence of a nuclearly-encoded mitochondrially-targeted
mRNA, such as the 3'UTR sequence of a naturally-occurring
nuclearly-encoded mitochondrially-targeted mRNA, or [0219] the cDNA
sequence of such a RNA, or [0220] a DNA sequence coding for such a
3'UTR sequence, or [0221] a conservative variant or fragment of
such a RNA or DNA 3'UTR sequence, which derives therefrom by
deletion and/or substitution and/or addition of one or several
nucleotides, and which, when replacing the wild-type 3'UTR of said
nuclearly-encoded mitochondrially-targeted mRNA, still allows for a
mitochondrial targeting of the resulting mRNA.
[0222] It may be provided that, when said at least one MTS nucleic
acid sequence is the MTS RNA sequence of a naturally-occurring
nuclearly-encoded mitochondrially-targeted mRNA, or the cDNA
sequence of such a mRNA, or a DNA sequence coding for such a MTS
RNA sequence in accordance with the universal genetic code, said at
least one nucleic acid CDS sequence is not the CDS of this
naturally-occurring nuclearly-encoded mitochondrially-targeted
mRNA.
[0223] It may be provided that, when said at least one 3' nucleic
acid sequence is the 3'UTR sequence of a naturally-occurring
nuclearly-encoded mitochondrially-targeted mRNA, or the cDNA
sequence of such a mRNA, or a DNA sequence coding for such a 3'UTR
sequence, said at least one CDS sequence is not the CDS of this
naturally-occurring nuclearly-encoded mitochondrially-targeted m
RNA.
[0224] It may be provided that, when said at least one MTS nucleic
acid sequence and said 3' nucleic acid sequence, respectively, are
the MTS and 3'UTR sequences of a naturally-occurring
nuclearly-encoded mitochondrially-targeted mRNA, or the cDNA
sequences of such a mRNA, or a DNA sequence coding for such a mRNA
sequence, then said at least one CDS sequence is not the CDS of
this naturally-occurring nuclearly-encoded mitochondrially-targeted
mRNA.
[0225] Preferably, said nucleic acid construct does not comprise
any sequence which would be identical to: [0226] the 3'UTR of a
naturally-occurring mRNA which is a nuclearly-transcribed but
not-mitochondrially-targeted mRNA, or [0227] the cDNA sequence of
such a 3'UTR sequence, or [0228] a DNA sequence coding for such a
naturally-occurring mRNA 3'UTR in accordance with the universal
genetic code.
[0229] The resulting nucleic acid construct does not use a
post-translation importation pathway, but uses a co-translation
importation pathway from nucleus to said mitochondrion.
[0230] The present invention more particularly relates to a
non-naturally occurring nucleic acid construct which comprises:
[0231] at least one mitochondrion-targeting nucleic acid sequence
(referred to as MTS nucleic acid sequence), [0232] at least one
nucleic acid sequence which encodes said protein in accordance with
the universal genetic code (referred to as CDS sequence), and
[0233] at least one 3' nucleic acid sequence, which is located in
3' of said at least one MTS nucleic acid sequence and of said at
least one CDS sequence, wherein said at least one MTS nucleic acid
sequence is: [0234] the cDNA sequence of the MTS RNA sequence of a
nuclearly-encoded mitochondrially-targeted mRNA, or [0235] a
conservative variant or fragment of such a cDNA sequence, which
derives therefrom by deletion and/or substitution and/or addition
of one or several nucleotides, but has retained a
mitochondrion-targeting function, wherein said at least one 3'
nucleic acid sequence is: [0236] the cDNA sequence of the 3'UTR
sequence of a nuclearly-encoded mitochondrially-targeted mRNA, or
[0237] a conservative variant or fragment of such a cDNA 3'UTR
sequence, which derives therefrom by deletion and/or substitution
and/or addition of one or several nucleotides, and which, when
replacing the wild-type 3'UTR of said nuclearly-encoded
mitochondrially-targeted mRNA, still allows for a mitochondrial
targeting of the resulting mRNA, provided that, when said at least
one MTS nucleic acid sequence and said at least one 3' nucleic acid
sequence, respectively, are the MTS and 3'UTR sequences of a
naturally-occurring nuclearly-encoded mitochondrially-targeted
mRNA, or the cDNA sequences of such a mRNA, or a DNA sequence
coding for such a mRNA sequence, then said at least one CDS
sequence is not the CDS of this naturally-occurring
nuclearly-encoded mitochondrially-targeted mRNA, and wherein said
nucleic acid construct does not comprise any sequence which would
be identical to: [0238] the 3'UTR of a naturally-occurring mRNA
which is a nuclearly-transcribed but not-mitochondrially-targeted
mRNA, or [0239] the cDNA sequence of such a 3'UTR sequence, or
[0240] a DNA sequence coding for such a naturally-occurring mRNA
3'UTR in accordance with the universal genetic code.
[0241] Each and every feature, herein and above described for the
MTS, CDS, 3' nucleic acid sequences in relation with the vector of
the invention, and notably those features relating to the MTS, CDS,
3' nucleic acid sequences, of course applies mutatis mutandis to
the nucleic acid contruct of the invention, and more particularly
to the non-naturally occurring nucleic acid contruct of the
invention.
[0242] Hence, it notably follows that: [0243] a MTS nucleic acid
sequence of said nucleic acid construct can be the MTS nucleic acid
sequence of ACO2, or of SOD2, or of ATP5b, or of UQCRFS1, or of
NDUFV1, or of NDUFV2, or of ALDH2, or of COX10; [0244] a 3' nucleic
acid sequence of said nucleic acid construct can be: [0245] the
3'UTR sequence of ACO2, or of SOD2, or of ATP5b, or of UQCRFS1, or
of NDUFV1, or of NDUFV2, or of ALDH2, or of COX10, or of AK2, or
[0246] the cDNA sequence of such a 3'UTR sequence, or [0247] a DNA
sequence coding for such a 3'UTR sequence in accordance with the
universal genetic code; and that [0248] illustrative nucleic acid
constructs of the invention comprise or consist of a sequence of
SEQ ID NO:21 (COX10 MTS-re-coded ATP6-COX10 3'UTR), and/or of SEQ
ID NO:22 (SOD2 MTS-re-coded ATP6-SOD2 3'UTR), and/or of SEQ ID
NO:25 (COX10 MTS-re-coded ND1-COX10 3'UTR), and/or of SEQ ID NO:26
(COX10 MTS-re-coded ND4-COX10 3'UTR).
[0249] Said non-naturally occurring nucleic acid construct may be
transfected in a cell in the form of naked DNA, or in the form of a
plasmid. Any transfection technology which is found convenient by
the skilled person is convenient. The skilled person may e.g.
proceed by electroporation, DEAE Dextran transfection, calcium
phosphate transfection, cationic liposome fusion, creation of an in
vivo electrical field, DNA-coated microprojectile bombardment, ex
vivo gene therapy, and the like.
[0250] Said non-naturally occurring nucleic acid construct may of
course alternatively and/or complementarily be inserted into a
vector, such as a viral vector.
[0251] The vector and nucleic acid construct of the invention are
useful for nucleic acid therapy, e.g. to reverse a cellular
dysfunction caused by a mutation in nucleic acid coding for a
mitochondrial protein. They enable to restore a protein function in
a cell.
Engineered Cell:
[0252] The present invention also relates to an engineered cell
which has been transduced or infected by a vector according to the
invention, and/or transfected by a nucleic acid construct according
to the invention.
[0253] Preferably, said engineered cell is an engineered animal or
human cell, most preferably to a mammalian engineered cell, still
more preferably to an engineered human cell.
[0254] Said engineered cell may e.g. be a bone-marrow cell, a
clonal cell, a germ-line cell, a post-mitotic cell, such as a cell
of the central nervous system; a neuronal cell, a retinal ganglion
cell, a progenitor cell; or a stem cell, a hematopoietic stem cell,
a mesenchymal stem cell. Preferably, said engineered cell is a
neuronal cell, a retinal ganglion cell.
[0255] Said transduction, infection or transfection may be impleted
by any means available to the skilled person, e.g. by
electroporation, DEAE Dextran transfection, calcium phosphate
transfection, cationic liposome fusion, creation of an in vivo
electrical field, DNA-coated microprojectile bombardment, injection
with a recombinant replicative-defective virus, homologous
recombination, ex vivo gene therapy, a viral vector, naked DNA
transfer, and the like.
[0256] Said engineered cell may e.g. be a cell, such as a neuronal
cell, collected from a patient suffering from a disease related to
a mitochondrial dysfunction. The vector and nucleic acid construct
of the invention can indeed be used for ex vivo cell therapy.
Pharmaceutical Compositions and Applications:
[0257] The present invention also relates to a pharmaceutical
composition comprising at least one vector according to the
invention, or at least one nucleic acid construct according to the
invention, or at least one engineered mammalian cell according to
the invention.
[0258] The present invention also relates to a drug comprising at
least one vector according to the invention, or at least one
nucleic acid construct according to the invention, or at least one
engineered mammalian cell according to the invention.
[0259] The compositions of the present invention may further
comprise at least one pharmaceutically and/or physiologically
acceptable vehicle (diluent, excipient, additive, pH adjuster,
emulsifier or dispersing agent, preservative, surfactant, gelling
agent, as well as buffering and other stabilizing and solubilizing
agent, etc.).
[0260] Appropriate pharmaceutically acceptable vehicles and
formulations include all known pharmaceutically acceptable vehicles
and formulations, such as those described in "Remington: The
Science and Practice of Pharmacy", 20.sup.th edition, Mack
Publishing Co.; and "Pharmaceutical Dosage Forms and Drug Delivery
Systems", Ansel, Popovich and Allen Jr., Lippincott Williams and
Wilkins.
[0261] In general, the nature of the vehicle will depend on the
particular mode of administration being employed. For instance,
parenteral formulations usually comprise, in addition to the one or
more contrast agents, injectable fluids that include
pharmaceutically and physiologically acceptable fluids, including
water, physiological saline, balanced salt solutions, buffers,
aqueous dextrose, glycerol, ethanol, sesame oil, combinations
thereof, or the like as a vehicle. The medium also may contain
conventional pharmaceutical adjunct materials such as, for example,
pharmaceutically acceptable salts to adjust the osmotic pressure,
buffers, preservatives and the like. The carrier and composition
can be sterile, and the formulation suits the mode of
administration.
[0262] The composition can be e.g., be in the form of a liquid
solution, suspension, emulsion, capsule, sustained release
formulation, or powder.
[0263] The pharmaceutical composition and drug of the invention are
useful for the therapeutic and/or palliative and/or preventive
treatment of a disease, condition, or disorder related to a defect
in activity or function of mitochondria.
[0264] A mitomap is available on the MITOMAP webiste; this site
notably provides with a list of mitochondrial disease-associated
mutations.
[0265] Scientific publication review relating to mitochondrial
disease, condition, or disorder notably comprise Carelli et al.
2004 (Progress in Retinal and Eye Research 23: 53-89), DiMauro 2004
(Biochimica et Biophysica Acta 1659:107-114), Zeviani and Carelli
2003 (Curr Opin Neurol 16:585-594), and Schaefer et al. 2004
(Biochimica et Biophysica Acta 1659: 115-120), the contents of
which being herein incorporated by reference.
[0266] Diseases, conditions or disorders related to a defect in
mitochondria activity or function notably comprise myopathies and
neuropathies, such as optic neuropathies.
[0267] Examples of mitochondrial diseases, conditions or disorders
comprise: aging, aminoglycoside-induced deafness, cardiomyopathy,
CPEO (chronic progressive external ophtalmoplegia),
encephalomyopathy, FBSN (familial bilateral stritial necrosis), KS
(Kearns-Sayre) syndrome, LHON (Leber's hereditary optic
neuropathy), MELAS (mitochondrial myopathy, encephalopathy, lactic
acidosis, and stroke-like episodes), MERRF (myoclonic epilepsy with
stroke-like episodes), MILS (maternally-inherited Leigh syndrome),
mitochondrial myopathy, NARP (neropathy, ataxia, and retinis
pigmentosa), PEO, SNE (subacute necrotizing encephalopathy).
[0268] Optic neuropathies notably comprise: [0269] Leber's
hereditary optic neuropathy (LHON), which involves one or several
point mutation(s) in mitochondrial DNA, more particularly point
mutation(s) in the ND1 and/or ND4 and/or ND6 gene(s), such as
G3460A (ND1 mutation), G11778A (ND4 mutation), T14484C (ND6
mutation), [0270] the dominant optic atrophy (DOA), also known as
Kjer's optic neuropathy, which involves a defect in nuclear gene
OPA1, [0271] the FBSN, MILS and NARP, which are the result of a
mutation in the MTATP6 gene (defective ATP synthesis), which can be
corrected by restoring the activity of function of ATP6.
[0272] Leber's hereditary optic neuropathy (LHON) was the first
maternally inherited disease to be associated with point mutations
in mitochondrial DNA and is now considered the most prevalent
mitochondrial disorder. The pathology is characterized by selective
loss of retinal ganglion cells leading to central vision loss and
optic atrophy, prevalently in young males. It is a devastating
disorder with the majority of patients showing no functional
improvement and remaining within the legal requirement for blind
registration. Other clinical abnormalities have also been reported
in LHON patients. These include postural tremor, peripheral
neuropathy, non-specific myopathy, movement disorders and cardiac
arrhythmias [8]. The three most common pathogenic mutations from
LHON affect complex I ND1 and/or ND4 and/or ND6 genes with the
double effect of lowering ATP synthesis and increasing oxidative
stress chronically.
[0273] Each of said disease, condition or disorder could be
corrected by restoring activity or function of the mutated DNA.
[0274] Example 1 below illustrates the rescue of an ATP6 activity
or function with a vector and nucleic acid of the invention.
Example 2 below illustrates the rescue of a ND1 and ND4 activity or
function with a vector and nucleic acid of the invention
(fibroblasts collected from LHON patients).
[0275] The pharmaceutical compositions or drugs of the invention
are more particularly intended for the therapeutic and/or
palliative and/or preventive treatment of a myopathy or of an optic
neuropathy, such as LHON, DOA, FBSN, MILS or NARP.
[0276] The present invention relates to the use of at least one
vector or nucleic acid construct for in vivo or ex vivo therapy of
a subject or patient in need of a therapeutic, palliative or
preventive treatment of a disease, condition, or disorder related
to a defect in activity or function of mitochondria.
[0277] The present invention also relates to the use of at least
one vector or nucleic acid construct of the invention, or of at
least one engineered cell of for the treatment of a disease,
condition, or disorder related to a defect in activity or function
of mitochondria, and more particularly for the production of a
composition, pharmaceutical composition or drug intended for the
treatment of such a disease condition, or disorder.
[0278] The present invention more particularly relates to a method
for the therapeutic and/or palliative and/or preventive treatment
of a disease, condition, or disorder related to a defect in
activity or function of mitochondria, which comprises: [0279]
administering to a subject or patient in need thereof a vector
and/or a nucleic acid construct and/or an engineered cell of the
invention, in a quantity effective for the therapeutic and/or
palliative and/or preventive treatment of said subject or patient,
[0280] ex vivo treating cells collected from a subject or patient
in need thereof, and returning the treated cells to the subject or
patient.
[0281] The term "comprising", which is synonymous with "including"
or "containing", is open-ended, and does not exclude additional,
unrecited element(s), ingredient(s) or method step(s), whereas the
term "consisting of" is a closed term, which excludes any
additional element, step, or ingredient which is not explicitly
recited.
[0282] The term "essentially consisting of" is a partially open
term, which does not exclude additional, unrecited element(s),
step(s), or ingredient(s), as long as these additional element(s),
step(s) or ingredient(s) do not materially affect the basic and
novel properties of the invention.
[0283] The term "comprising" (or "comprise(s)") hence includes the
term "consisting of" ("consist(s) of"), as well as the term
"essentially consisting of" ("essentially consist(s) of").
Accordingly, the term "comprising" (or "comprise(s)") is, in the
present application, meant as more particularly encompassing the
term "consisting of" ("consist(s) of"), and the term "essentially
consisting of" ("essentially consist(s) of").
[0284] Each of the relevant disclosures of all references cited
herein is specifically incorporated by reference.
[0285] The present invention is illustrated by the following
examples, which are given for illustrative purposes only.
EXAMPLES
Example 1
Allotopic Expression of the ATP6 Mitochondrial Gene is
Significantly Improved by the Localization of its mRNAs to the
Surface of Mitochondria Leading to an Efficient Import of the
Precursor
Abstract:
[0286] It is clear that impairment of mitochondrial energy
metabolism is the key pathogenic factor in a growing number of
neurodegenerative disorders. With the discovery of mtDNA mutations,
the replacement of defective genes became an important goal for
mitochondrial geneticists worldwide. Unfortunately, before the
present invention, it was still not possible to introduce foreign
genes into the mitochondria of mammalian cells.
[0287] To circumvent this problem, allotopic expression in the
nucleus of genes encoded by mitochondrial DNA (mtDNA), became an
attractive idea. However, for most mitochondrial genes tested,
there were important limitations related to the high hydrophobicity
of the corresponding proteins, which impedes their mitochondrial
translocation.
[0288] We herein elucidate the mechanisms that enable the delivery
of mRNAs encoding mitochondrial proteins to the organelle surface,
and demonstrate that this delivery depends on two sequences: the
region coding for the mitochondrial targeting sequence (MTS) and
the 3'UTR. mRNA sorting to mitochondrial surface permits to
optimize allotopic approach, by enhancing the mitochondrial import
efficiency of the precursor synthesized in the cytosol. As an
illustration of this mechanism, we have chosen to utilize the
sequence coding for the MTS and the 3'UTR of two nuclear genes
encoding mitochondrial proteins: COX10 and SOD2 associated to a
recoded mitochondrial ATP6 gene. Indeed, COX10 and SOD2 mRNAs
localize to the mitochondrial surface in HeLa cells. HeLa cells
transfected with these constructions express an Atp6 protein which
is successfully delivered to the mitochondria. Hence, we have been
able to optimize the allotopic approach for Atp6, and our procedure
will next be tried to rescue mitochondrial dysfunction in patients
presenting ATP6 mutations.
Introduction
[0289] To examine the possibility that allotopic expression of
mtDNA genes could be optimized by a targeted localization of the
mRNA to the mitochondrial surface, we have chosen to utilize the
sequences coding for the MTS and the 3'UTR of two nuclear genes
encoding mitochondrial proteins: COX10 and SOD2 associated to an
reengineered nucleus-localized ATP6 gene. COX10 encodes a highly
hydrophobic protein of the inner mitochondrial membrane, its mRNAs
localizes to the mitochondrial surface [5]. SOD2 encodes a
mitochondrial protein involved in detoxification, its mRNA, as
COX10 mRNA, localizes to the mitochondrial surface [5] and a recent
report described that in HeLa cells, its 3'UTR is associated to the
mitochondrial surface via the Akap121 protein [6]. The ability to
synthesize and direct the Atp6 protein to mitochondria was examined
in Hela cells for 4 plasmids: two of them only contain the mts of
COX10 or SOD2, and the two other possess both the MTS and the 3'UTR
of each gene. Hybrid mRNAs were detected for each construction in
both transiently and stably transfected cells. Further, Atp6
protein was also visualized by indirect immunofluorescence
associated to the surface of mitochondria. Mitochondria isolated
from transfected cells were examined for the presence of Atp6
protein. Remarkably, hybrids mRNAs possessing both the MTS and the
3'UTR of either COX10 and SOD2 allow the synthesis of a polypeptide
which is imported in a highly efficient way from the cytosol into
the mitochondria. Thus, the strategy of directing a hybrid mRNA to
the mitochondrial surface significantly improves the feasibility of
the allotopic approach for mitochondrial genes.
Material and Methods:
[0290] Plasmid construction: The full-length ATP6 mitochondrial
gene was reengineered after the production of the 677 pb product by
RT-PCR (Superscript III one step RT-PCR Platinium Taq HiFi,
Invitrogen), using total RNA from HeLa cells. The PCR product
obtained was cloned in the PCR 2.1-Topo vector (Invitrogen, Life
technologies). In this vector, we recoded 11 non-universal codons
in the ATP6 gene by four rounds of in vitro mutagenesis (Quik
change Multi site-directed mutagenesis kit; Stratagene, La Jolla,
Calif.). Six oligonucleotide primers were designed to alter AUA
codons to AUG and UGA to UGG (Table 1).
TABLE-US-00006 TABLE 1 In vitro mutagenesis of the ATP6
mitochondrial gene Length Name sequence (bp) ATP6.1
CAATGGCTAATCAAACTAACCTCAAAACAAATGATG 78
ACCATGCACAACACTAAAGGACGAACCTGGTCTCT TATGCTA(SEQ ID NO: 1) ATP6.2
TCTATGAACCTAGCCATGGCCATCCCCTTATGGGCG 54 GGCACAGTGATTATGGGC (SEQ ID
NO: 2) ATP6.3 CCCATGCTAGTTATTATCGAAACCATCAGCCTACTCA 51
TTCAACCAATGGCC (SEQ ID NO: 3) ATP6.4 ACCCTAGCAATGTCAACCATTAAC 24
(SEQ ID NO: 4) ATP6.5 ACTAAAGGACGAACCTGGTCTCTTATGCTAGTATC 42
CTTAATC (SEQ ID NO: 5) ATP6.6 ACACCAACCACCCAACTATCTATGAACCTAGCCAT
42 GGCCATC (SEQ ID NO: 6)
[0291] The intermediate construct was sequenced for accuracy. To
this recoded ATP6, we appended in frame either the MTS of COX10 or
SOD2, obtained by RT-PCR using total RNA from HeLa cells
(Superscript III one step RT-PCR Platinium Taq HiFi; Invitrogen,
Life technologies). For COX10 we amplified the sequence
corresponding to the first 28 amino acids, for SOD2 the sequence
coding for the first 30 amino acids. Oligonucleotide primers used
for the amplification include at its 3' extremity a SalI
restriction site for the subsequent cloning in frame with the
reengineered ATP6 gene which possesses a SalI restriction site at
its 5' extremity (Table 2).
TABLE-US-00007 TABLE 2 Oligonucleotides primers for RT-PCR analysis
RT-PCR product length Name 5' Primer (5'-3') 3'Primer (5'-3') (bp)
ATP6 ORF GTCGACCGCATGA CCGGGCGGCCGCTGT 677 ACGAAAATCTGTTC
GTTGTCGTGCAGGTA GCTTCATTCATT GAGGCTTAC (SEQ ID NO: 7) (SEQ ID NO:
8) MTS CGCTCTAGAATGG GCGGTCGACTTCAAG 84 COX10 CCGCATCTCCGCA
ATACCAGACAGAGCC CACTCTC TCC (SEQ ID NO: 9) (SEQ ID NO: 10) 'UTR
CCCGATCGGAGCA CGCACGCGTAAAGCT 1429 COX10 CTGGGACGCCCAC
TCTACAAATGTGAAGG CGCCCCTTTCCC CTGTAACA (SEQ ID NO: 11) (SEQ ID NO:
12) MTS SOD2 CGCTCTAGAATGTT GTCGACCGCGTCGGG 90 GAGCCGGGCAGTG
GAGGCTGTGCTTCTG TGCGGC CCT (SEQ ID NO: 13) (SEQ ID NO: 14) 3'UTR
ACCACGATCGTTAT CGCACGCGTCAATCA 215 SOD2 GCTGAGTATGTTAA
CACAAAGCATTTACTA GCTCTTTA TTTTC (SEQ ID NO: 15) (SEQ ID NO: 16)
COX6c ATGGCTCCCGAAG CTGAAAGATACCAGC 250 TTTTGCCAAAACCT CTTCCTCATCTC
(SEQ ID NO: 17) (SEQ ID NO: 18) SOD2 CGACTACGGCGCC CTGGAACCTCACA
TCAACGC (SEQ ID NO: 58)
[0292] The final sequences of the fusion ATP6 genes were checked
for accuracy, and inserted in the pCMV-Tag 4A vector (Stratagene,
La Jolla Calif.), which will direct the synthesis of the protein
via the CMV promoter and its detection by the presence of a FLAG
epitope tag appended to the C-terminal region of Atp6. To obtain
hybrid mRNAs which will also contain the 3'UTR of COX10 or SOD2
genes we replaced the SV40 polyA signal present in the pCMV-Tag 4A
vector (positions 1373-1679) using Pvu1 and Mlu1 restriction
enzymes, by the 1429 by of the full-length COX10 3'UTR or the 215
pb of the SOD2 3'UTR. Both 3'UTR were first obtained by RT-PCR
using RNAs purified from HeLa cells and specific oligonucleotide
primers containing Pvu1 and Mlu1 restriction sites at each end (cf.
Table 2 above). PCR fragments were first cloned in the PCR 2.1-Topo
vector (Invitrogen, Life technologies) and sequenced to verify that
no mistakes were generated before subcloning in the pCMV-Tag 4A
vector. The four final constructs were entirely sequenced for
accuracy using specific oligonucleotide primers to verify the
full-length sequences of either the fusion ATP6 genes or the 3'UTR
regions appended to them. Final sequences inserted in the pCMV-Tag
4A vectors are shown in FIG. 1B.
[0293] The sequence of recoded ATP6 (SEQ ID NO: 27) is shown on
FIG. 9. The MTS and 3'UTR sequences of COX10 and SOD2 are shown on
FIG. 10 (SEQ ID NO: 30, 47, 31 and 60).
Cell Culture and Transfection:
[0294] We cultured HeLa cells with RPMI medium complemented with
10% of foetal bovine serum (Gibco, Invitrogen), gentamicin (0.01%),
2 mM glutamine, optionally with pyruvate (e.g., 2.5 mM), optionally
with antibiotics (such as 100 u/mL penicillin, 100 .mu.g/mL
streptomycin). They were transfected with FuGENE 6 transfection
reagent as recommended by the manufacturer (Roche Biochemicals,
Indianapolis). Briefly, monolayer Hela cells were seeded a day
before transfection at 50% confluence, so the next day they will be
at approximately 80% confluence, the cells were plated in a medium
without antibiotics. 2 microgrammes of different plasmids purified
with Quiagen plasmid midi kit (Quiagen; Valencia, Calif.) were
used. Between 48 to 60 hr later, 80% of the transfected cells were
used either for immunochemistry analyses or RNA and mitochondria
extractions. The remaining 20% of cells were selected for
neomycine, G418, resistance (selectable marker present in the
pCMV-Tag 4A vector) at a final concentration of 1 mg/ml. Stable
clones were expanded for several weeks, immunochemistry analyses
were performed. Mitochondria were also isolated to determine the
import ability of the Atp6 protein.
[0295] Immunocytochemistry:
[0296] Coverslips were placed on the bottom of 24-well dishes and
HeLa cells seeded at approximately 50% confluence (80000 cells). 60
hours after transfection, cells were fixed with 2% paraformaldehyde
in PBS for 15 min and processed for indirect immunofluorescence.
After permeabilization of the cells for 5 min with Triton 1% in
PBS, cells were incubated for one hour in PBS with 1% BSA before
the addition of the primary antibodies: mouse monoclonal anti-Flag
M2 antibodies (Stratagene, La Jolla Calif.) or mouse monoclonal
anti-ATP synthase subunit beta (Molecular Probes, Invitrogen). Both
antibodies were used at a final concentration of 1 microgramme/ml.
The incubation with primary antibodies was performed for either 2
hr at room temperature or overnight at 4.degree. C. After washing
the primary antibody three times five min with PBS, cells were
incubated with the secondary antibody: labeled
goat-anti-mouse.quadrature.IgG Alexa Fluor 488 (Molecular Probes,
Invitrogen). This antibody was used in 1% BSA-PBS at 1:600 dilution
and placed on the top of the coverslips for two hr. The cells were,
subsequently, washed once in PBS for 5 min. For DNA and
mitochondria staining, a second wash was performed with 0.3
microgrammes/ml of DAPI (Sigma, Saint Louis, Mich.) and 100 nM of
MitoTracker Deep Red 633 (Molecular probes, Invitrogen) for 20 min.
A last 10 min wash was performed in PBS and the coverslips were
mounted using Biomeda Gel/Mount. Immunofluorescence was visualized
in a Leica DM 5000 B Digital Microscope. Digital images were
acquired and processed with the MetaVue imaging system
software.
[0297] Mitochondria Isolation and Western Blot Analysis:
[0298] Between 20 to 40 millions or 100 millions of transiently or
stably transfected HeLa cells were treated with trypsin (Gifco,
Invitrogen) for 5 min and spin down. One wash in PBS was performed.
The pellets were resuspended in 10 ml of homogenization buffer: 0.6
Mannitol, 30 mM Tris-HCl PH 7.6, 5 mM MgAc and 100 mM KCl, 0.1%
fatty acid-free bovine serum albumin (BSA), 5 mM
beta-mercaptoethanol and 1 mM PMFS. To the resuspended cells 0.01%
of digitonin was added. After a 4 min incubation on ice the
homogenization was performed with 15 strokes in a Dounce glass
homogenenizer with a manually driven glass pestle type B.
Homogenates are centrifuged for 8 min at 1000 g at 4.degree. C. to
pellet unbroken cells and nuclei. Since many mitochondria remain
trapped in this pellet, it was resuspended and rehomogenized again
with 5 ml of homogenization buffer and 25 additional strokes. Then
a second round of centrifugation under the same conditions was
performed. Both supernatants were assembled and centrifuged again
to discard any nuclear or cell contaminant. The supernatant
obtained was centrifuged at 12000 g at 4.degree. C. for 30 min to
pellet mitochondria. Four washes in homogenization buffer were
performed to free the mitochondrial fraction of particles
containing membranes, reticulum endoplasmic and proteases. The last
two washes were performed in a homogenization buffer devoid of BSA
and PMFS to allow a better estimation of the protein concentration
in the final mitochondrial fraction and its subsequent analysis by
proteinase K digestion. Protein concentrations in the extracts were
measured using the dye-binding assay Bradford. To determine whether
Atp6 was translocated into the organelle, 15 microgrammes of
mitochondrial proteins were treated with 200 microgrammes/ml of
proteinase K (PK) at 0.degree. C. for 30 min. Samples were then
resolved in 4-12 gradient or 12% polyacrylamide SDS-PAGE, and
transferred to nitrocellulose. Filters were probed with the
following antibodies: mouse monoclonal anti-Flag M2 antibodies
(Stratagene, La Jolla Calif.) which recognizes the nuclear recoded
Atp6 protein in which a flag epitope was appended at its C-terminus
or mouse monoclonal anti-ATP synthase subunit alpha (Molecular
Probes, Invitrogen), which recognizes the 65 kDa nuclear encoded
alpha-subunit of the ATP synthase, Complex V. Immunoreactive bands
were visualized with anti-mouse coupled to horseradish peroxidase
(1:10000) followed by ECL Plus detection (Amersham International)
according to the manufacturer's instructions.
[0299] Five independent mitochondria purifications from cells
stably transfected with either SOD2.sup.MTS ATP6-3'UTR.sup.SV40 or
SOD2.sup.MTS ATP6-3'UTR.sup.SOD2 vectors were performed. The amount
of precursor and mature forms of ATP6 in mitochondria, as well as
the quantities of both the mature form of ATP6 and ATP .quadrature.
resistant to PK proteolysis were compared by densitometric analyses
(Quantity One, Biorad software system). The significance of the
differences observed was validated with a paired Student's
t-test.
RNA Extraction and RT-PCR Analyses:
[0300] Mitochondria extractions were performed as described in the
precedent section, with the following modifications: 400 millions
of cells were treated with 250 .mu.g/ml cycloheximide for 20
minutes at 37.degree. C. To HB was added 200 Mg/ml cycloheximide,
500 .mu.g/ml heparine and 1/1000 RNase inhibitor (rRNasin,
Promega). The last pellet of crude mitochondria associated with
polysomes (M-P) was stored at -80.degree. C. until RNA extraction.
Free-cytoplasmic polysomes (F-P) were obtained from the
post-mitochondrial supernatant fraction by sedimentation through a
step gradient of 2 M and 0.5 M sucrose. RNAs from these two
fractions, as well as total RNAs from each stably transfected cell
line, were obtained using RNeasy Protect Mini kit (Qiagen).
Generally, 10 millions of cells are sufficient to obtain
approximately 30 microgrammes of total RNA. The presence of the
hybrid ATP6 mRNA was examined using primers which recognize the
first 27 nt of either COX10 or SOD2 MTS and a primer which
recognize the last 27 nt of the ATP6 ORF. For the pCMV-Tag 4A
vector containing both the MTS and the 3'UTR of COX10 or SOD2, we
used a primer recognizing the last 27 nt of each 3'UTR. 100 ng of
RNA was used for reverse transcription (cf. Table 1 above). The
products were then subjected to 25 cycles of PCR using Superscript
III one step RT-PCR Platinium Taq kit (Invitrogen). As an internal
control a 250 nt fragment within the ORF of COX6c gene, encoding a
mitochondrial protein, was also amplified. Ten percent of the
amplified products were run in agarose gels, and the quantities of
amplified products reflecting hybrid ATP6 mRNA amount in each
preparation was estimated using the Photocap software (Vilber
Lourmat; Torcy, France).
[0301] Tables 1 and 2 above, and table 9 below, show primer
sequences, the expected sizes of the PCR products, the quantity of
RNA used for reverse-transcription and the number of PCR cycles
performed.
[0302] Densitometric analyses (Quantity One, Bio-Rad software) were
performed the amount of both hybrid ATP6 and SOD2 transcripts in
either mitochondrion-bound polysomes or free-cytoplasmic polysomes.
Three independent RNA preparations from M-P and F-P fractions were
subjected three times to RT-PCR analyses.
TABLE-US-00008 TABLE 9 RT- PCR Polysomal RNAs product Primers Total
RNAs (M-P/F-P) length 5' Quantity Cycle Quantity Cycle mRNA (bp)
Primer 3' Primer (ng) numbers (ng) numbers SOD2.sup.MTS 780 MTS
ATP6 200 28 250 28 ATP6 SOD2 5' ORF3' ATP6 677 ATP6 ATP6 50 28 150
20 ORF 5' ORF 3' SOD2 785 SOD2 5' 3'UTR 100 28 20 20 SOD2 3' COX6c
250 COX6c COX6c 3' 200 28 250 20 5'
Results:
Construction of Reengineered Mitochondrial ATP6 Gene for Allotopic
Expression
[0303] To accomplish allotopic expression we synthesized the
full-length version of nuclear-encoded ATP6 mitochondrial gene
converting codons AUA to AUG and codons UGA to UGG. Indeed, AUA in
the mitochondrial genetic system leads to the insertion of a
methionine, but according to the universal code it is an
isoleucine. Additionally, UGA into mitochondria codes for a
tryptophan, whereas in the cytosol it represents a stop codon. We,
therefore, recoded all 11 mitochondrial codons present in ATP6
ensuring the accurate translation of the transcript by cytoplasmic
ribosomes. These alterations were performed by four rounds of in
vitro mutagenesis using six independent oligonucleotide primers
(Table 1) and the Quik change Multi site-directed mutagenesis kit
(Stratagene, La Jolla, Calif.).
[0304] The concept of allotopic approach has important implications
for the development of therapies to patients with mitochondrial DNA
mutations. However, up today a major obstacle remains to be
overcome and is the targeting of the recoded protein to
mitochondria. We then decided to force the localization of the
recoded ATP6 mRNA to the mitochondrial surface. The rationale
behind this specific mRNA targeting is to allow a co-translational
import mechanism which will maintain the precursor in an import
competent conformation impeding its aggregation before or during
translocation through the TOM (Translocase of the outer membrane)
and TIM (Translocases of the inner membrane) import complexes. Two
sequences within mRNAs are believed to be involved in their
localization to the mitochondrial membrane: the sequence coding for
the MTS and the 3'UTR. We have chosen two nuclearly-encoded
mitochondrial genes, which mRNAs are preferentially localized to
the surface of mitochondria in HeLa cells: COX10 and SOD2 [5].
Interestingly, the SOD2 mRNA has been shown to be associated to the
mitochondrial surface via its 3'UTR and the Akap121 protein.
[0305] We therefore, obtained four different plasmids.
[0306] Two contain either the MTS of COX10 or the sequence encoding
the first 30 amino acids of SOD2 (=the 20 amino acids of the MTS
sequence of SOD2, and the ten consecutive amino acids that follows
within the SOD2 sequence, i.e., fragment 1-30 of SEQ ID NO:49), in
frame with the AUG codon of the recoded ATP6 gene (COX10
MTS-recoded ATP6-SV40 3' UTR; SOD2 MTS-recoded ATP6-SV40 3' UTR).
In these plasmids, the SV40 polyA signal functions as the
3'UTR.
[0307] The other two combine both the MTS and the 3'UTR of COX10
and SOD2 respectively, and do not comprise the cytosolic 3'UTR of
SV40 (COX10 MTS-recoded ATP6-COX10 3' UTR; SOD2 MTS-recoded
ATP6-SOD2 3' UTR).
[0308] FIGS. 1A and 1B illustrate the constructs obtained and the
full-length sequences inserted in the pCMV-Tag 4A vector than we
named respectively: COX10 MTS-nATP6, SOD2 MTS-nATP6 and COX10
MTS-nATP6-COX10 3'UTR and SOD2 MTS-nATP6-SOD2 3'UTR.
Detection of Hybrid ATP6 mRNAs in Transiently and Stably HeLa
Transfected Cells
[0309] To determine whether transfected cells express hybrid ATP6
mRNAs, steady-state levels of the transcripts were measured in both
transiently and stably transfected cells after the isolation of
total RNAs. 100 ng of total RNAs were subjected to RT-PCR analyses
using specific primer oligonucleotides for hybrid ATP6 mRNA. COX6c
gene encoding a mitochondrial protein was used as an internal
control, with specific primers allowed the amplification of a 250
by fragment (FIG. 2). RNAs from non-transfected HeLa cells as well
as HeLa cells transfected with the empty pCMV-Tag 4A vector were
also tested as negative controls. FIG. 2 shows a 780 by PCR product
corresponding to the amplification of the first 27 nt of COX10 ORF
and the last 27 nt of the ATP6 ORF in cells transfected with both
COX10 MTS-nATP6 and COX10 MTS-nATP6-COX10 3'UTR vectors.
Additionally, RNAs isolated from cells transfected with the COX10
MTS-nATP6-COX10 3'UTR vector amplified a 2374 by product
corresponding to the entire ATP6 ORF and the full-length COX10
3'UTR.
[0310] The results obtained with RNAs purified from transfected
cells with SOD2 MTS-nATP6 and SOD2 MTS-nATP6-SOD2 3'UTR vectors
show that hybrid ATP6 transcript was detected as a 780 nt amplified
product. Further, the SOD2 MTS-nATP6-SOD2 3'UTR region amplified a
1060 by fragment, corresponding to the entire ATP6 ORF and the
full-length SOD2 3'UTR. These results indicate that HeLa cells
express the reengineered ATP6 gene. Moreover, no significant
differences in the steady-state levels of hybrid mRNAs were found
by the addition of either COX10 3'UTR or SOD2 3'UTR.
[0311] To examine the ability of SOD2 signals associated to the
recoded ATP6 gene to direct hybrid mRNAs to the mitochondrial
surface, we determined their subcellular localization in the four
stably cell lines obtained. In this purpose, we isolated RNAs from
mitochondrion-bound polysomes (M-P) and free-cytoplasmic polysomes
(F-P) and we determined by RT-PCR the steady-state levels of hybrid
mRNAs in both polysomal populations (FIG. 12B). As internal
controls, the subcellular distribution of endogenous mitochondrial
ATP6, SOD2 and COX6c mRNAs were determined. Endogenous ATP6 mRNA
exclusively localized to the mitochondrial compartment as expected.
Besides, endogenous SOD2 mRNA is enriched in mitochondrion-bound
polysomes (M-P), whereas COX6c mRNA is preferentially detected in
free-cytoplasmic polysomes (F-P) as we have previously observed.
The SOD2MTSATP6-3'UTRSOD2 vector directed the synthesis of a hybrid
mRNA that was almost undetectable in free-cytoplasmic polysomes.
Hybrid mRNA produced from the SOD2MTSATP6-3'UTRSV40 plasmid was
also detected preferentially in mitochondrion-bound polysomes.
However, it was also present in free-cytoplasmic polysomes (FIG.
12B). Densitometric analyses were performed to determine the amount
of both endogenous SOD2 and hybrid ATP6 mRNAs in each polysomal
population examined. SOD2 mRNA signal in mitochondrion-bound
polysomes was 85.6%.+-.6.15 in cell lines expressing the
SOD2MTSATP6-3'UTRSV40 plasmid and 82.5%.+-.4.87 in cells expressing
the SOD2MTSATP6-3'UTRSOD2 vector. Interestingly, it was found for
the ATP6 hybrid mRNA that only 72.4%.+-.5.2 localized to the
mitochondrial surface in cells expressing the SOD2MTSATP6-3'UTRSV40
vector. Instead, in cells expressing the SOD2MTSATP6-3'UTRSOD2
vector 84.6%.+-.4.7 of the hybrid mRNA localized to the
mitochondrial surface (FIG. 12C). These values were significantly
different according to the paired Student's t-test (P<0.0034,
n=6). Thus, the combination of both the MTS and 3'UTR of SOD2 to
the reengineered ATP6 gene leads to the synthesis in the nucleus of
a transcript which was almost exclusively sorted to the
mitochondrial surface. Indeed, its subcellular distribution is not
significantly different to the one of the endogenous SOD2 mRNA.
TABLE-US-00009 TABLE 10 ATP6 mRNA signal localized at the ATP6 mRNA
within Hela cells mitochondrial surface the mitochondria With a
cytosolic 3'UTR 72.4% .+-. 5 0.71 .+-. 0.12 (SV40 3' UTR) With a
mitochondrial .sup. 82.5 .+-. 4.8 1.28 .+-. 0.24 3'UTR (SOD2
3'UTR), and without any cytosolic 3'UTR
Detection of ATP6 Allotopic Expression in HeLa Cells by Indirect
Immunofluorescence
[0312] We analyzed the ability of the reengineered ATP6 product to
localize to mitochondria in vivo.
[0313] For this, we appended a Flag epitope in frame to the
C-terminus of the ATP6 ORF and we examined stably transfected cells
by indirect immunofluorescence (FIG. 13). HeLa cells transfected
with the empty pCMV-Tag 4A vector were used as negative controls
and showed a low diffused signal in cytoplasm when antibodies to
Flag were used (FIG. 13, left panel). Stably transfected cells with
either SOD2.sup.MTS ATP6-3'UTR.sup.SV40 or SOD2.sup.MTS
ATP6-3'UTR.sup.SOD2 vectors were visualized by indirect
immunofluorescence using antibodies to Flag (FIG. 14, left panel)
and to ATP synthase subunit .alpha. (FIG. 13 middle panel). For
each cell type visualized, a merged image in association with DAPI
staining is shown in the right panel. A typical punctuate
mitochondrial pattern was observed in cells expressing the recoded
ATP6 polypeptides, when the Flag antibody was used. This indicates
that fusion ATP6 proteins localized to mitochondria.
[0314] Immunocytochemistry to detect the flag epitope in HeLa cells
transiently or stably transfected with the four pCMV-Tag 4A vectors
showed a typical punctuate mitochondrial pattern, suggesting that
the fusion Atp6 protein had been localized within the mitochondria
(FIG. 3). Indeed, this typical punctuate mitochondrial pattern was
also observed using either the mitochondrion-specific dye Mito
Tracker Red or specific antibodies anti-ATP synthase subunit beta.
HeLa cells transfected with the empty pCMV-Tag 4A vector were used
as negative controls and showed a diffuse cytoplasmic distribution
but with a low intensity (FIG. 3). The localization patterns of the
different Atp6 peptides which synthesis were directed by the four
pCMV-Tag 4A vectors were essentially identical confirming that both
COX10 and SOD2 sequences successfully allowed the reengineered Atp6
protein to localize to the mitochondria in vivo.
Translocation of the Fusion Atp6 Protein into Mitochondria of HeLa
Cells
[0315] To determine whether reengineered ATP6 gene products are
efficiently imported into mitochondria in vivo, mitochondria
isolated from stably transfected HeLa cells were subjected to
western blot analysis (FIG. 4). We visualized two forms with
anti-flag antibodies of approximately 30 and 20 kDa, representing
the precursor and mature forms of the recoded ATP6 protein.
[0316] The predicted molecular weights of both proteins are
respectively 34 and 30 kDa, larger than the ones implied by the
molecular weight markers. This discrepancy has often been observed
when extremely hydrophobic proteins were migrated in SDS-PAGE. In
general, the electrophoretic mobility on SDS-PAGE of proteins
encoded by mtDNA is higher than the one expected for their
theoretical molecular weights.
[0317] The steady-state levels of both polypeptides are similar in
the two cell lines examined: cells transfected with MTS COX10-nATP6
vector (MTS COX10-nATP6), and cells transfected with MTS
COX10-nATP6-COX10 3'UTR (MTS COX10-nATP6-3'UTR).
[0318] To determine the amounts of the recoded ATP6 polypeptides
produced in HeLa cells expressing either SOD2.sup.MTS
ATP6-3'UTR.sup.SV40 or SOD2.sup.MTS ATP6-3'UTR.sup.SOD2 vectors, we
compared six independent mitochondrial extractions (FIG. 14A). Both
precursor and mature polypeptides were equally abundant in
mitochondria from each cell line, indeed the expression of
SOD2.sup.MTS ATP6-3'UTR.sup.SV40 vector leads to an accumulation of
61.4%.+-.6 of the precursor form. Instead, SOD2.sup.MTS
ATP6-3'UTR.sup.SOD2 vector directed the synthesis of 64.4%.+-.6.5
of the precursor. These values were not significantly different
according to the paired Student's t-test. Similar results were
obtained when total extracts from each cell line were examined by
Western blotting. This data is in agreement with the overall
amounts of hybrid ATP6 mRNAs detected when total RNAs from cell
lines expressing either SOD2.sup.MTS ATP6-3'UTR.sup.SV40 or
SOD2.sup.MTS ATP6-3'UTR.sup.SOD2 vectors were subjected to RT-PCR
analyses (FIG. 12A). Therefore, the steady-state levels of the ATP6
precursor and its ability to recognize the TOM complex in the outer
mitochondrial membrane do not depend on the presence of the SOD2
3'UTR. Notably, the relative proportions of ATP6 precursor and
mature forms were analogous to the ones shown in cells for highly
hydrophobic proteins en route to the mitochondria.
[0319] When mitochondria were treated with 150 or 200
microgrammes/ml of proteinase K (PK) the precursor forms of the
fusion ATP6 protein were sensitive to proteolysis in both cells
lines. In contrast the mature form of ATP6 is resistant to PK
digestion, especially in cells expressing the MTS COX10-nATP6-COX10
3'UTR transcript. Indeed, in these cells the amount of the mature
ATP6 protein is approximately 185% higher than in cells expressing
the MTS COX10-nATP6 mRNA. These data strongly indicates that not
only the precursor polypeptide is correctly addressed to the
surface of mitochondria, as we observed by indirect
immunofluorescence (FIG. 3), but also that it was efficiently
translocated into the organelle and correctly processed. Moreover,
FIG. 4 shows that the quantity of the mature form of the Atp6
protein and the 65 kDa ATPalpha protein inside the mitochondria
were quite similar after proteinase K digestion. Therefore, the use
of COX10 MTS allows an efficient mitochondrial translocation of the
recoded ATP6 protein, and when COX10 MTS is combined to the 3'UTR
of COX10 a significant more efficient in vivo translation/import of
the allotopically expressed ATP6 gene is obtained.
[0320] FIG. 14B upper panel shows a schematic representation of the
theoretically expected ATP6 import intermediate. The hydrophobic
passenger ATP6 precursor can be trapped en route to the matrix and
a mitochondrial processing peptidase can cleave the MTS.
Nevertheless, the rest of the protein remained accessible to PK
action and therefore becoming undetectable on Western blotting.
Instead, the fraction of the ATP6 protein which can be completely
translocated is insensitive to PK-induced proteolysis and can
therefore be integrated into the inner mitochondrial membrane,
hence, remaining detectable on immunoblotting.
[0321] FIG. 14B shows that precursor forms of the fusion proteins
were sensitive to proteolysis in both cell line examined. Nearly
all the ATP6 precursor signal disappeared after PK digestion, so
precursors that were engaged in the process of translocation or
loosely attached to the outer mitochondrial membrane but not fully
translocated, were entirely digested (FIG. 14B, middle panel). In
contrast, a significant amount of the mature form of ATP6 is
resistant to PK digestion, indicating its location inside the
organelle. To examine the levels of another complex V protein in
these cells, immunoblots were performed using anti-ATP synthase
.alpha. antibody. This naturally imported mitochondrial protein was
present at similar extents in all cells tested. Only one band of
approximately 65 kDa was visualized suggesting that either we were
unable to discriminate the precursor and mature forms of this
protein under the electrophoretic conditions used or precursor
polypeptides were very rapidly and efficiently translocated.
Additionally, no major differences of the ATP synthase .alpha.
signals were detected after PK treatment, confirming the integrity
of the mitochondrial isolations (FIG. 14B, middle panel). To
compare the import efficiency of the recoded ATP6 proteins in cells
transfected with either SOD2.sup.MTS ATP6-3'UTR.sup.SV40 or
SOD2.sup.MTS ATP6-3'UTR.sup.SOD2 vectors, we measured the amount of
the mature form of ATP6 insensitive to PK digestion in each cell
line, after normalization with the amount of ATP synthase .alpha.
resistant to PK proteolysis. Results for six independent
mitochondrial extractions subjected to immunoblotting analyses were
shown in FIG. 15B, lower panel. Overall results show that both SOD2
signals lead to a high efficient import of the recoded ATP6
precursor. Remarkably, the level of the mature form insensitive to
PK proteolysis in cells transfected with SOD2.sup.MTS
ATP6-3'UTR.sup.SOD2 (1.28.+-.0.24) was 1.8 fold higher than in
cells expressing the SOD2.sup.MTS ATP6-3'UTR.sup.SV40
(0.71.+-.0.12). The difference measured was significant according
to the paired Student's t-test (P<0.0022, n=6). This observation
could be related to the higher enrichment in the
mitochondrion-bound polysomes of the corresponding mRNA (FIG.
12B).
[0322] The question arises whether imported reengineered ATP6
proteins were assembled into the ATP synthase complex. The complex
is organized in F0-F1 domains, F1 sector is a water-soluble unit
located in the matrix and having the ability to hydrolyse ATP. The
F0 domain is embedded in the inner membrane and is composed by
hydrophobic subunits forming a proton pathway. ATP6 is an intrinsic
protein of F0, composed of five putative transmembranous
.alpha.-helices. In contrast, ATP synthase .alpha. is a located in
the matrix F1 domain. Studies performed with bovine heart
mitochondria demonstrated that ATP6 was degraded at a very low rate
when F0 subunits were subjected to trypsine treatment. Therefore,
we treated mitochondria with both PK and Triton X-100 (1%). The
detergent disrupts both mitochondrial membranes and theoretically
leads to the entire proteolysis of mitochondrial proteins,
demonstrating their localization somewhere inside the organelle in
a protease-sensitive form. FIG. 3C shows that indeed ATP synthase
.alpha. was fully digested by PK; instead a significant amount of
ATP6 remained insensitive to PK proteolysis. This result suggests
that the recoded ATP6 was assembled into complex V.
Discussion
[0323] Recent, epidemiological studies demonstrated that as a
group, disorders of the mitochondrial function affect at least 1 in
5000 of the population, making them among the most common
genetically determined disorders. In spite of the fact that over
the last decade, the underlying genetic bases of several
mitochondrial diseases involving central nervous system
degeneration, no effective therapy is available for mitochondrial
disorders. Pathogenic point mutations of genes encoded by the
mitochondrial genome have been described as the cause of many
mitochondrial disorders. A possible therapeutic approach is
therefore to exploit the natural mitochondrial protein import
pathway. The basic concept is to introduce a wild-type copy of the
mutated mitochondrial gene into the nucleus and import normal
copies of the gene product into mitochondria from cytosol. This
concept has been termed allotopic expression and several reports in
yeast described that a number of non mitochondrial polypeptides can
be relocated to the mitochondrial matrix simply by conjugating a
targeting sequence to their N-terminus. However, when this approach
has been tried in mammalian cells using different MTS and genes
encoded by mtDNA, precursors were not imported efficiently into
mitochondria. By consequence, the rescue of mitochondrial defect in
patient cells was not only partial but also temporary [7].
Therefore, up today the spectrum of mtDNA encoded polypeptides than
can be successfully expressed and integrated into mitochondrial
respiratory chain complexes is very limited. This limitation is
thought to be the consequence of the high hydrophobicity nature of
mtDNA encoded proteins, which possess transmembrane domains
refractive to mitochondrial import. The precursor synthesized in
the cytosol could lack the import-competent structure required for
an efficient mitochondrial membrane translocation.
[0324] The concept of mesohydrophobicity is likely to be an
important factor for mitochondrial import competency.
Mesohydrophobicity describes the average hydrophobicity in a window
of 60-80 amino acids, together with the calculation of the most
hydrophobic 17-amino acid segment. This calculation could predict
importability of hydrophobic peptides. Using their algorithm, we
analyzed this correlation to assess the mitochondrial importability
of SOD2.sup.MTS ATP6 gene product and compared to ATP6, COX8 and
SOD2 polypeptides as well as the previously tested fusion protein
COX8.sup.MTSATP6: as the wild-type ATP6, both fusion proteins
examined cannot be translocated into mitochondria, mainly due to
the high hydrophobicity of ATP6. Hence, a possibility that can
allow the import of a recoded ATP6 protein into the organelle is
that the precursor is engaged in a co-translational pathway of
import. Thereby, the precursor would be maintained in a loosely
folded nonaggregated conformation required for translocation
through the mitochondrial import apparatus.
[0325] To overcome this limitation and try to develop a more
long-term and definitive rescue of mtDNA mutations by allotopic
expression leading to its application in gene therapy, we decided
to construct nuclear versions of the mtDNA encoded ATP6 gene in
which we appended the signals intended for forcing the hybrid mRNA
to localize to the mitochondrial surface. We have chosen COX10 and
SOD2 genes, which transcripts are enriched in the mitochondrial
surface.
[0326] We were able to demonstrate that the association to a
recoded ATP6 gene of both the MTS and 3'UTR signals leading to a
mRNA delivery to the mitochondrial surface unambiguously improves
the feasibility of the allotopic approach for mitochondrial genes.
Indeed, not only we were able to visualize the protein in the
mitochondria by indirect immunofluorescence, but most important the
amounts of the processed Atp6 polypeptide inside the organelle were
quite similar to the naturally imported ATPalpha protein. This
result strongly indicates that the recoded Atp6 precursor was
efficiently imported, the improvement we were able to produce as
compared to other recent reports [1], [2], [3], [4] is certainly
due to the fact that the hybrid mRNA was addressed to the
mitochondrial surface, therefore enhancing the coupling between
translation and mitochondrial import processes.
[0327] Most interestingly, we obtained a gradual improvement,
indeed the use of either COX10 or SOD2 MTS alone, gave a good
result in which approximately 50% of the mature ATP6 protein is
translocated inside the mitochondria. When each MTS was combined to
the corresponding 3'UTR, at least 85% of the mature ATP6 protein is
insensitive to proteinase K digestion indicating that almost all
the protein synthesized in the cytoplasm is successfully
translocated inside the mitochondria.
[0328] To accomplish allotopic expression, the localization of an
mRNA to the mitochondrial surface has never been tried before. In
the allotopic approaches reported, even though different MTS were
appended to recoded mitochondrial genes, all the constructs
examined contained at their 3'extremities the SV40 polyA signal
that does not lead to any specific subcellular localization of the
transcript. Our data clearly demonstrate that the association to a
recoded ATP6 gene of both the MTS and 3'UTR signals of the SOD2
gene leads to a high efficient delivery of the hybrid mRNA to the
mitochondrial surface. This improves unambiguously the feasibility
of the allotopic approach for this mitochondrial gene. Indeed, not
only were we able to visualize ATP6 protein in the mitochondria by
indirect immunofluorescence but definitely the amount of the
processed ATP6 polypeptide inside the organelle was quite similar
to the naturally imported ATP synthase subunit .alpha., a Complex V
component, such as is ATP6. These data strongly indicate that
recoded ATP6 precursors were successfully imported. The improvement
we obtained compared to a recent report, which measured 18.5% of
the precursor translocated, is certainly due to the localization of
hybrid mRNAs to the mitochondrial surface. This specific
localization obviously enhances the coupling between translation
and import processes, therefore, diminishing the block of the
precursor during its translocation through the TOM and TIM
complexes. It is worth mentioning that we obtained a gradual
improvement on mitochondrial import of the ATP6 precursor. When
both the MTS and the 3'UTR of SOD2 were combined, the amount of
fully translocated ATP6 protein was 1.8-fold higher than when just
the MTS was present. This is likely related to the improvement of
mRNA sorting to the mitochondrial surface when both cis-acting
elements of SOD2 were associated to the recoded ATP6 gene.
Remarkably, proteolysis insensitivity of the translocated ATP6
protein in the presence of both PK and Triton X-100 suggested that
the protein could be correctly assembled in the F0 domain of the
respiratory chain Complex V. Notably, combining the cis-acting
elements of the COX10 gene to the recoded ATP6 gene, we obtained a
very efficient mitochondrial import ability of the fusion protein.
Indeed, COX10 mRNA codes for a highly hydrophobic protein involved
in Complex IV biogenesis, and SOD2 mRNA is enriched at the
mitochondrial surface in human cells.
[0329] We clearly demonstrated that the association to a recoded
ATP6 gene of both the MTS and 3'UTR signals of either SOD2 or COX10
genes leads to a high efficient delivery of hybrid ATP6 mRNAs to
the mitochondrial surface, especially when both the MTS and the
3'UTR of SOD2 or COX10 were associated to the reengineered ATP6
gene. This specific subcellular localization of hybrid mRNAs leads
to a high efficiency in the mitochondrial translocation of the
recoded ATP6 proteins. Remarkably, when both the MTS and the 3'UTR
of either SOD2 or COX10 were combined, the amount of fully
translocated ATP6 protein was 1.8-fold higher than when the MTS was
associated to the cytosolic SV40 3'UTR. Therefore, the improvement
of mRNA sorting to the mitochondrial surface when both cis-acting
elements of SOD2 or COX10 were associated to the recoded ATP6 gene
definitely increase the amount of the processed ATP6 polypeptide
inside the organelle which became quite similar to the naturally
imported ATP synthase subunit .alpha., a Complex V component, such
as is ATP6. Thus, by directing a hybrid mRNA to the mitochondrial
surface we significantly improve the feasibility of the allotopic
approach for the ATP6 mitochondrial gene.
[0330] In conclusion, we optimize the allotopic expression approach
for ATP6, by the use of mRNA targeting signals without any amino
acid change in the protein that could affect biologic activity.
[0331] This approach becomes henceforth available to rescue
mitochondrial deficiencies caused by mutations in mtDNA genes.
Example 2
Correct Mitochondrial Localization of the Recoded Mitochondrial ND1
and ND4 Genes in Fibroblastes from LHON Patients
[0332] The three most common pathogenic mutations from LHON affect
complex I ND1, ND4 and ND6 genes with the double effect of lowering
ATP synthesis and increasing oxidative stress chronically.
[0333] Since we have demonstrated that reengineered mitochondrial
Atp6 proteins were successfully translocated inside the
mitochondria in HeLa cells (see example 1 above), we decided to
synthesize recoded mitochondrial genes ND1 and ND4. To ensure the
efficient import of the allotopically expressed proteins we
appended to them signals which will direct the corresponding mRNAs
to the mitochondrial surface. We have chosen to use the MTS of
COX10 gene alone or in combination with its entire 3'UTR. FIGS. 5A
and 5B illustrate the constructs obtained and the full-length
sequences inserted in the pCMV-Tag 4A vector.
Material and Methods:
[0334] Cell Culture and Transfection:
[0335] Fibroblasts were obtained from LHON patients of the Hopital
Necker Enfants Malades, Paris, France (Departement de Genetique).
We cultured these cells with D-MEM medium complemented with 10% of
foetal bovine serum, pyruvate, gentamicin (0.01%), and 2 mM
glutamine. When indicated cells were grown in glucose-free medium
supplemented with 10 mM galactose.
[0336] Fibroblasts were transfected with FuGENE 6 transfection
reagent as recommended by the manufacturer (Roche Biochemicals,
Indianapolis). Briefly, monolayer fibroblast cells were seeded a
day before transfection at 50% confluence, so the next day they
will be at approximately 80% confluence, the cells were plated in a
medium without antibiotics. 2 microgrammes of different plasmids
purified with Quiagen plasmid midi kit (Quiagen; Valencia, Calif.)
were used. Between 48 to 60 hr later, 80% of the transfected cells
were used for immunochemistry analyses. The remaining 20% of cells
were selected for neomycine, G418, resistance (selectable marker
present in the pCMV-Tag 4A vector) at a final concentration of 0.25
mg/ml. Stable clones were expanded for several weeks.
Optimized Recoding into Human Genetic Code
[0337] mtDNA has been recoded according to human genetic code,
taking into account the preferred codon usage in human:
TABLE-US-00010 TABLE 3 preferred codon usage in human Source Human
preferred codon usage ARG CGA -- CGC -- CGG -- CGU -- AGA -- AGG
AGG LEU CUA -- CUC -- CUG CUG CUU -- UUA -- UUG -- SER UCA -- UCC
UCC UCG -- UCU -- AGC -- AGU -- THR ACA -- ACC ACC ACG -- ACU --
PRO CCA -- CCC CCC CCG -- CCU -- ALA GCA -- GCC GCC GCG -- GCU --
GLY GGA -- GGC GGC GGG -- GGU -- VAL GUA -- GUC -- GUG GUG GUU --
LYS AAA -- AAG AAG ASN AAC AAC AAU -- GLN CAA -- CAG CAG HIS CAC
CAC CAU -- GLU GAA -- GAG GAG ASP GAC GAC GAU -- TYR UAC UAC UAU --
CYS UGC UGC UGU -- PHE UUC UUC UUU -- ILE AUA -- AUC AUC AUU -- MET
AUG AUG TRP UGG UGG % GC 63/42
ND1 and ND4 Constructs:
[0338] COX10 MTS-nND1-SV40 3' UTR, COX10 MTS-nND4-SV40 3' UTR,
COX10 MTS-nND1-COX10 3'UTR, and COX10 MTS-nND4-COX10 3'UTR were
produced as described above in example 1 for ATP6. The resulting
sequences are shown on FIG. 5B (SEQ ID NO:23, 24, 25, 26,
respectively).
Results:
[0339] Detection of ND1 Allotopic Expression in Fibroblasts from
LHON Patients Presenting the G3460A ND1 Mutation
[0340] We analyzed the ability of the reengineered ND1 product to
localize to mitochondria in vivo. Immunocytochemistry analyses were
performed to detect the flag epitope in fibroblasts, from a patient
presenting the ND1 gene mutated, transiently transfected with
either COX10 MTS-nND1-SV40 3' UTR or COX10 MTS-nND1-COX10 3'UTR
showed a typical punctuate mitochondrial pattern, suggesting that
the fusion Nd1 protein had been localized within the mitochondria
(FIG. 6). Indeed, this typical punctuate mitochondrial pattern was
also observed using specific antibodies anti-ATP synthase subunit
beta. Cells transfected with the empty pCMV-Tag 4A vector were used
as negative controls and showed a diffuse cytoplasmic distribution
but with a low intensity (FIG. 6). The localization patterns of Nd1
peptides which synthesis were directed by the two vectors examined
were essentially identical confirming that COX10 sequences
successfully allowed the reengineered Nd1 protein to be addressed
inside the mitochondria.
Detection of ND4 Allotopic Expression in Fibroblasts from LHON
Patients Presenting the G11778A ND4 Mutation
[0341] Two plasmids directing the synthesis in the cytosol of a
recoded wild-type ND4 gene were obtained. One of them, COX10
MTS-nND4-SV40 3' UTR, possesses appended to the N-terminus of the
protein the sequence corresponding to the first 28 amino acids of
Cox10. The second one, COX10 MTS-nND4-COX10 3'UTR, has in addition
at the end of the ORF the full-length 3'UTR of COX10. Fibroblasts
from a patient presenting 100% of mtDNA molecules with the G11778A
ND4 mutation were transiently transfected with either one of these
plasmids. 60 h later cells were fixed and visualized to determine
the ability of the COX10 sequences to target the recoded protein to
the mitochondria. FIG. 7 shows that in both cases the fusion MTS
Cox10ND4Flag protein did have a punctuate staining pattern, which
is very similar to the one observed for the same cells with the
naturally imported mitochondrial protein ATP synthase subunit beta.
Thus, implying that the recoded Nd4 fusion protein was imported
into mitochondria.
[0342] In conclusion, as for the mitochondrial ATP6 gene, we were
able to optimize the allotopic expression approach for ND1 and ND4
genes, by the simply use of mRNA targeting signals without any
amino acid change in the protein that could affect biologic
activity.
Growth Ability of LHON Fibroblasts in Galactose Medium
[0343] Fibroblasts presenting the G3460A ND1 mutation were grown
with galactose, which slowly enters glycolysis as compared to
glucose. FIG. 8 shows major differences in cell growth after six
day culture: fibroblasts presented a severe growth defect, less
that 10% of the cells survived in medium containing galactose as
compared to cells seeded in glucose-rich medium. Stably transfected
fibroblasts with the MTS COX10-nND1-COX10 3'UTR vector had a
markedly improved rate of growth in galactose compared with that of
non-transfected cells. This result implies that the mitochondrially
imported recoded ND1 protein had assembled successfully into
functional complex I allowing, therefore, a rescue of mitochondrial
dysfunction in these cells.
Example 3
Rescue of Mitochondrial Deficiency Causing Human Diseases
(Transfection of Fibroblasts from a NARP Patient)
[0344] We also determined whether the reengineered ATP6 protein
would be able to rescue mitochondrial deficiency in cells having a
mutated ATP6 gene.
[0345] We obtained fibroblasts from a patient presenting NARP
disease caused by the T8993G mutation in the ATP6 gene.
[0346] Fibroblasts were cultured on media containing sodium
pyruvate and relatively high amounts of FBS, more particularly:
[0347] on a medium containing glucose (D-MEM with L-glutamine, 4500
mg/L D-glucose, 110 mg/L sodium pyruvate 2.5 mM, FBS 15%, uridine
28 microM), or [0348] on a medium, which does not contain glucose,
but contain galactose (liquid D-MEM (1.times.), with L-Glutamine
without Glucose, sodium pyruvate 2.5 mM, galactose 10 mM, FBS 15%,
uridine 28 microM).
[0349] Stably transfected cells expressing the nuclear version of
ATP6 associated with either SOD2 MTS alone, or in combination with
SOD2 3'UTR, were obtained. Respiratory chain activity has been
examined by the ability of these cells to grow in a medium in which
glucose has been replaced by galactose for either 10 or 20 days.
NARP cells expressing the empty vector had a low survival rate
(30%). Cells expressing ATP6 with either the MTS of SOD2 or both
the MTS and the 3'UTR of SOD2 present a growth survival of
approximately 60%. If the selection was maintained for 20 days, the
survival growth rate of cells expressing our optimized vectors was
superior to 80% (FIG. 16). Only subtle differences of survival rate
were observed for fibroblasts expressing either the vector with
both the MTS and 3'UTR of the SOD2 gene or the vector with the SOD2
MTS associated to the SV40 3'UTR. This, is certainly due to the
fact that these cells are heteroplasmic for the T8993G mutation,
indeed they possess approximately 10% of the wild-type gene.
Therefore, in their mitochondria probably 10% of a functional ATP6
protein could be assembled in Complex V. We can envision that the
expression of the vector with SOD2 MTS associated to the SV40
3'UTR, will lead to the mitochondrial import of enough ATP6 protein
to allow the cells to growth a good rate in galactose medium.
[0350] Preliminary measures of the real amount of ATP produced in
vitro by fibroblasts expressing either ones of our vectors clearly
show a difference in the activity of Complex V related to the
presence of either SOD2 3'UTR or SV40 3'UTR. Hence, when compared
to control fibroblasts (100% of ATP synthesis in galactose medium)
NARP fibroblasts expressing the vector with SOD2 MTS associated to
the SV40 3'UTR had 50%, representing an increase compared to non
transfected NARP cells (30%) but was less important when compared
to the amount found in cells expressing the vector which combines
to the recoded ATP6 gene both the MTS and the 3'UTR of the SOD2
gene (approximately 85%); cf. FIG. 19. By consequence, a more
complete and efficient rescue of mitochondrial dysfunction is
obtained when allotopic approach implies the presence of both the
MTS and 3'UTR targeting signals.
TABLE-US-00011 TABLE 11 Survival rate Rate of ATP synthesis
Fibroblasts on galactose on galactose Control 100% 100% NARP
(mutated ATP6) 30% 30% NARP + cytosolic 3'UTR 60% 50% (SV40 3'UTR)
NARP + mitochondrial 60% 85% 3'UTR (SOD2 3'UTR)
Example 4
Rescue of Mitochondrial Deficiency Causing Human Diseases
(Transfection of Fibroblasts from LHON Patients)
[0351] The applicability potential of the improved allotopic
expression approach of the inventors has been further confirmed by
examining two other mtDNA genes involved in LHON. The fibroblasts
obtained presented a total homoplasmy of the mutation; indeed all
the molecules of mitochondrial DNA are mutated.
[0352] Fibroblasts were cultured on media containing sodium
pyruvate and relatively high amounts of FBS, more particularly:
[0353] on a medium containing glucose (D-MEM with L-glutamine, 4500
mg/L D-glucose, 110 mg/L sodium pyruvate 2.5 mM, FBS 15%, uridine
28 microM), or [0354] on a medium, which does not contain glucose,
but contain galactose (liquid D-MEM (1.times.), with L-Glutamine
without Glucose, sodium pyruvate 2.5 mM, galactose 10 mM, FBS 15%,
uridine 28 microM).
[0355] The engineered nucleus-localized versions of ND1 and ND4
were obtained; ND1 and ND4 transcripts possess both at their 5' and
3' extremities COX10 mRNA targeting sequences. Stable transfections
of these constructions in fibroblasts from LHON's patients with
either ND1 or ND4 mutations were performed. Indirect
immunofluorescence showed that both proteins localize to the
surface of mitochondria in vivo. The OXPHOS activity of these cells
has been also examined by growing in a galactose rich medium.
Interestingly, fibroblast cells allotopically expressing the
wild-type ND4 protein showed a markedly improved rate of growth on
galactose medium. This improvement is higher when both the MTS and
3'UTR of COX10 were associated to the ND4 gene (54.3%) as compared
to that of mock transfected cells (8%) or to the cells transfected
with the ND4 gene associated to the MTS of COX10 and the cytosolic
SV40 3'UTR (12.7%) (FIG. 17, MTS: ND4 associated to COX10 MTS and
the SV40 3'UTR, 3'UTR: ND4 associated to both the MTS and 3'UTR of
COX10). This data imply that in spite of the presence in these
cells of the ND4 mutated polypeptide, the wild-type protein was
successfully imported into the organelle and assembled in Complex
I. Preliminary experiments, of in vitro measurements of ATP
synthesis confirm these results, indeed in untransfected cells very
little ATP was synthesized in galactose medium (14% of the control
level measured in healthy fibroblasts), when cells express the ND4
gene associated to the MTS of COX10 and the cytosolic SV40 3'UTR an
increased is observed (56%). Remarkably, this increase is more
important when cells express the ND4 gene associated to both the
MTS and 3'UTR of COX10 (84%).
[0356] Hence, our results undeniably confirm that we have optimized
the allotopic approach for three mtDNA encoded genes by the use of
mRNA targeting signals, without any amino acid change in the
proteins. This is particularly the case when our vectors presented
both the MTS and the 3'UTR targeting signals of a mRNA which
exclusively localized to the mitochondrial surface.
TABLE-US-00012 TABLE 12 Survival rate Rate of ATP synthesis
Fibroblasts on galactose on galactose Control 100% 100% LHON
(mutated ND4) 8% 14% LHON + cytosolic 3'UTR 12.7% 56% (SV40 3'UTR)
LHON + mitochondrial 54.3% 84% 3'UTR (COX10 3'UTR)
Example 5
Transduction of Retinal Ganglion Cells
[0357] The inventors obtained, by in vitro mutagenesis,
reengineered ND1, ND4, ND6 and ATP6 genes, which possess the most
common mutations found in LHON's and NARP's patients: G3460A,
G11778A, T14484C and T8993G respectively. Both wild-type and
mutated genes have been integrated in the p-AAV-IRES-hrGFP vector,
which will allow the production of infectious recombinant human
Adeno-Associated Virus Type 2 (AAV2) virions. For all
constructions, each nuclear version of mtDNA genes is associated to
the two mRNA targeting sequences of the COX10 gene, which allow the
enrichment of corresponding mRNAs at the surface of mitochondria.
In accordance with the present invention, this will ensure the
efficient delivery of the polypeptides inside the organelle.
[0358] Retinal ganglion cells (RGC) represent the primary cellular
target of the pathogenic process of LHON disease.
[0359] The inventors purified RGCs from adult rat retina, thereby
obtaining enriched RGC populations, and maintained them in culture
for two weeks. Mitochondria are distributed along actin filaments
and they specifically concentrated at the extremities of neuron
extensions. The inventors transfected these cells with the mutated
version of the ND1 gene. Preliminary results showed that the
expression at high levels of the mutated protein during 8 days
leads to an abnormal distribution of mitochondria along the neurite
and cone extensions (FIG. 18).
BIBLIOGRAPHIC REFERENCES CITED IN THE EXAMPLES
[0360] 1. Owen, R., et al., Recombinant Adeno-associated virus
vector-based gene transfer for defects in oxidative metabolism.
Hum. Gene Ther., 2000. 11: p. 2067-2078. [0361] 2. Guy, J., et al.,
Rescue of a mitochondrial deficiency causing Leber Hereditary Optic
Neuropathy. Ann. Neurol., 2002. 52: p. 534-542. [0362] 3. Manfredi,
G., et al., Rescue of a deficiency in ATP synthesis by transfer of
MTATP6, a mitochondrial DNA-encoded gene to the nucleus. Nature
Genet., 2002. 30: p. 394-399. [0363] 4. Oca-Cossio, J., et al.,
Limitations of allotopic expression of mitochondrial genes in
mammalian cells. Genetics, 2003. 165: p. 707-720. [0364] 5.
Sylvestre, J., et al., The role of the 3'UTR in mRNA sorting to the
vicinity of mitochondria is conserved from yeast to human cells.
Mol. Biol. Cell, 2003. 14: p. 3848-3856. [0365] 6. Ginsberg, M. D.,
et al., PKA-dependent binding of mRNA to the mitochondrial AKAP121
protein. J. Mol. Biol., 2003. 327(4): p. 885-897. [0366] 7. Smith,
P. M., et al., Strategies for treating disorders of the
mitochondrial genome. Biochem. Biophys. Acta, 2004. 1659: p.
232-239. [0367] 8. Carelli et al., Progress in Retinal and Eye
Research, 2004. 23: p. 53-89
Sequence CWU 1
1
60178DNAArtificial SequenceSynthetic Primer 1caatggctaa tcaaactaac
ctcaaaacaa atgatgacca tgcacaacac taaaggacga 60acctggtctc ttatgcta
78254DNAArtificial SequenceSynthetic Primer 2tctatgaacc tagccatggc
catcccctta tgggcgggca cagtgattat gggc 54351DNAArtificial
SequenceSynthetic Primer 3cccatgctag ttattatcga aaccatcagc
ctactcattc aaccaatggc c 51424DNAArtificial SequenceSynthetic Primer
4accctagcaa tgtcaaccat taac 24542DNAArtificial SequenceSynthetic
Primer 5actaaaggac gaacctggtc tcttatgcta gtatccttaa tc
42642DNAArtificial SequenceSynthetic Primer 6acaccaacca cccaactatc
tatgaaccta gccatggcca tc 42739DNAArtificial SequenceSynthetic
Primer 7gtcgaccgca tgaacgaaaa tctgttcgct tcattcatt
39839DNAArtificial SequenceSynthetic Primer 8ccgggcggcc gctgtgttgt
cgtgcaggta gaggcttac 39933DNAArtificial SequenceSynthetic Primer
9cgctctagaa tggccgcatc tccgcacact ctc 331033DNAArtificial
SequenceSynthetic Primer 10gcggtcgact tcaagatacc agacagagcc tcc
331138DNAArtificial SequenceSynthetic Primer 11cccgatcgga
gcactgggac gcccaccgcc cctttccc 381239DNAArtificial
SequenceSynthetic Primer 12cgcacgcgta aagcttctac aaatgtgaag
gctgtaaca 391333DNAArtificial SequenceSynthetic Primer 13cgctctagaa
tgttgagccg ggcagtgtgc ggc 331433DNAArtificial SequenceSynthetic
Primer 14gtcgaccgcg tcggggaggc tgtgcttctg cct 331536DNAArtificial
SequenceSynthetic Primer 15accacgatcg ttatgctgag tatgttaagc tcttta
361636DNAArtificial SequenceSynthetic Primer 16cgcacgcgtc
aatcacacaa agcatttact attttc 361727DNAArtificial SequenceSynthetic
Primer 17atggctcccg aagttttgcc aaaacct 271827DNAArtificial
SequenceSynthetic Primer 18ctgaaagata ccagccttcc tcatctc
27191337DNAArtificial SequenceSynthetic COX10 MTS-nATP6
19gaattcgccc ttcgctctag aatggccgca tctccgcaca ctctctcctc acgcctcctg
60acaggttgcg taggaggctc tgtctggtat cttgaagtcg accgcatgaa cgaaaatctg
120ttcgcttcat tcattgcccc cacaatccta ggcctacccg ccgcagtact
gatcattcta 180tttccccctc tattgatccc cacctccaaa tatctcatca
acaaccgact aatcaccacc 240caacaatggc taatcaaact aacctcaaaa
caaatgatga ccatgcacaa cactaaagga 300cgaacctggt ctcttatgct
agtatcctta atcattttta ttgccacaac taacctcctc 360ggactcctgc
ctcactcatt tacaccaacc acccaactat ctatgaacct agccatggcc
420atccccttat gggcgggcac agtgattatg ggctttcgct ctaagattaa
aaatgcccta 480gcccacttct taccacaagg cacacctaca ccccttatcc
ccatgctagt tattatcgaa 540accatcagcc tactcattca accaatggcc
ctggccgtac gcctaaccgc taacattact 600gcaggccacc tactcatgca
cctaattgga agcgccaccc tagcaatgtc aaccattaac 660cttccctcta
cacttatcat cttcacaatt ctaattctac tgactatcct agaaatcgct
720gtcgccttaa tccaagccta cgttttcaca cttctagtaa gcctctacct
gcacgacaac 780acagcggccg cccggaaggg cgaattcgat atcaagctta
tcgataccgt cgacctcgag 840gattacaagg atgacgacga taagtagggc
ccggtacctt aattaattaa ggtaccaggt 900aagtgtaccc aattcgccct
atagtgagtc gtattacaat tcactcgatc gcccttccca 960acagttgcgc
agcctgaatg gcgaatggag atccaatttt taagtgtata atgtgttaaa
1020ctactgattc taattgtttg tgtattttag attcacagtc ccaaggctca
tttcaggccc 1080ctcagtcctc acagtctgtt catgatcata atcagccata
ccacatttgt agaggtttta 1140cttgctttaa aaaacctccc acacctcccc
ctgaacctga aacataaaat gaatgcaatt 1200gttgttgtta acttgtttat
tgcagcttat aatggttaca aataaagcaa tagcatcaca 1260aatttcacaa
ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
1320aatgtatctt aacgcgt 1337201356DNAArtificial SequenceSynthetic
SOD2MTS-nATP6 20gaattcgccc ttcgctctag aatgttgagc cgggcagtgt
gcggcaccag caggcagctg 60gctccggttt tggggtatct gggctccagg cagaagcaca
gcctccccga cgcggtcgac 120cgcatgaacg aaaatctgtt cgcttcattc
attgccccca caatcctagg cctacccgcc 180gcagtactga tcattctatt
tccccctcta ttgatcccca cctccaaata tctcatcaac 240aaccgactaa
tcaccaccca acaatggcta atcaaactaa cctcaaaaca aatgatgacc
300atgcacaaca ctaaaggacg aacctggtct cttatgctag tatccttaat
catttttatt 360gccacaacta acctcctcgg actcctgcct cactcattta
caccaaccac ccaactatct 420atgaacctag ccatggccat ccccttatgg
gcgggcacag tgattatggg ctttcgctct 480aagattaaaa atgccctagc
ccacttctta ccacaaggca cacctacacc ccttatcccc 540atgctagtta
ttatcgaaac catcagccta ctcattcaac caatggccct ggccgtacgc
600ctaaccgcta acattactgc aggccaccta ctcatgcacc taattggaag
cgccacccta 660gcaatgtcaa ccattaacct tccctctaca cttatcatct
tcacaattct aattctactg 720actatcctag aaatcgctgt cgccttaatc
caagcctacg ttttcacact tctagtaagc 780ctctacctgc acgacaacac
agcggccgcc cggtaagggc gaattcgata tcaagcttat 840cgataccgtc
gacctcgagg attacaagga tgacgacgat aagtagggcc cggtacctta
900attaattaag gtaccaggta agtgtaccca attcgcccta tagtgagtcg
tattacaatt 960cactcgatcg cccttcccaa cagttgcgca gcctgaatgg
cgaatggaga tccaattttt 1020aagtgtataa tgtgttaaac tactgattct
aattgtttgt gtattttaga ttcacagtcc 1080caaggctcat ttcaggcccc
tcagtcctca cagtctgttc atgatcataa tcagccatac 1140cacatttgta
gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa
1200acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata
atggttacaa 1260ataaagcaat agcatcacaa atttcacaaa taaagcattt
ttttcactgc attctagttg 1320tggtttgtcc aaactcatca atgtatctta acgcgt
1356212381DNAArtificial SequenceSynthetic COX10MTS-nATP6-COX10
3'UTR 21gaattcgccc ttcgctctag aatggccgca tctccgcaca ctctctcctc
acgcctcctg 60acaggttgcg taggaggctc tgtctggtat cttgaagtcg accgcatgaa
cgaaaatctg 120ttcgcttcat tcattgcccc cacaatccta ggcctacccg
ccgcagtact gatcattcta 180tttccccctc tattgatccc cacctccaaa
tatctcatca acaaccgact aatcaccacc 240caacaatggc taatcaaact
aacctcaaaa caaatgatga ccatgcacaa cactaaagga 300cgaacctggt
ctcttatgct agtatcctta atcattttta ttgccacaac taacctcctc
360ggactcctgc ctcactcatt tacaccaacc acccaactat ctatgaacct
agccatggcc 420atccccttat gggcgggcac agtgattatg ggctttcgct
ctaagattaa aaatgcccta 480gcccacttct taccacaagg cacacctaca
ccccttatcc ccatgctagt tattatcgaa 540accatcagcc tactcattca
accaatggcc ctggccgtac gcctaaccgc taacattact 600gcaggccacc
tactcatgca cctaattgga agcgccaccc tagcaatgtc aaccattaac
660cttccctcta cacttatcat cttcacaatt ctaattctac tgactatcct
agaaatcgct 720gtcgccttaa tccaagccta cgttttcaca cttctagtaa
gcctctacct gcacgacaac 780acagcggccg cccggaaggg cgaattcgat
atcaagctta tcgataccgt cgacctcgag 840gattacaagg atgacgacga
taagtagggc ccggtacctt aattaattaa ggtaccaggt 900aagtgtaccc
aattcgccct atagtgagtc gtattacaat tcactcgatc ggagcactgg
960gacgcccacc gcccctttcc ctccgctgcc aggcgagcat gttgtggtaa
ttctggaaca 1020caagaagaga aattgctggg tttagaacaa gattataaac
gaattcggtg cccagtgatc 1080acttgacagt tttttttttt tttaaatatt
acccaaaatg ctccccaaat aagaaatgca 1140tcagctcagt cagtgaatac
aaaaaaggaa ttatttttcc ctttgagggt ctttatacat 1200ctctcctcca
accccaccct ctattctgtt tcttcctcct cacatggggg tacacataca
1260cagcttcctc ttttggttcc atccttacca ccacaccaca cgcacactcc
acatgcccag 1320cagagtggca cttggtggcc agaaagtgtg agcctcatga
tctgctgtct gtagttctgt 1380gagctcaggt ccctcaaagg cctcggagca
cccccttcct ggtgactgag ccagggcctg 1440catttttggt tttccccacc
ccacacattc tcaaccatag tccttctaac aataccaata 1500gctaggaccc
ggctgctgtg cactgggact ggggattcca catgtttgcc ttgggagtct
1560caagctggac tgccagcccc tgtcctccct tcacccccat tgcgtatgag
catttcagaa 1620ctccaaggag tcacaggcat ctttatagtt cacgttaaca
tatagacact gttggaagca 1680gttccttcta aaagggtagc cctggactta
ataccagccg gatacctctg gcccccaccc 1740cattactgta cctctggagt
cactactgtg ggtcgccact cctctgctac acagcacggc 1800tttttcaagg
ctgtattgag aagggaagtt aggaagaagg gtgtgctggg ctaaccagcc
1860cacagagctc acattcctgt cccttgggtg aaaaatacat gtccatcctg
atatctcctg 1920aattcagaaa ttagcctcca catgtgcaat ggctttaaga
gccagaagca gggttctggg 1980aattttgcaa gttatcctgt ggccaggtgt
ggtctcggtt accaaatacg gttacctgca 2040gctttttagt cctttgtgct
cccacgggtc tgcagagtcc catctgccca aaggtcttga 2100agcttgacag
gatgttttca ttactcagtc tcccagggca ctgctggtcc gtagggattc
2160attggtcggg gtgggagagt taaacaacat ttaaacagag ttctctcaaa
aatgtctaaa 2220gggattgtag gtagataaca tccaatcact gtttgcactt
atctgaaatc ttccctcttg 2280gctgccccca ggtatttact gtggagaaca
ttgcatagga atgtctggaa aaagctccta 2340caacttgtta cagccttcac
atttgtagaa gctttacgcg t 2381221179DNAArtificial SequenceSynthetic
SOD2MTS-nATP6-SOD2 3'UTR 22gaattcgccc ttcgctctag aatgttgagc
cgggcagtgt gcggcaccag caggcagctg 60gctccggttt tggggtatct gggctccagg
cagaagcaca gcctccccga cgcggtcgac 120cgcatgaacg aaaatctgtt
cgcttcattc attgccccca caatcctagg cctacccgcc 180gcagtactga
tcattctatt tccccctcta ttgatcccca cctccaaata tctcatcaac
240aaccgactaa tcaccaccca acaatggcta atcaaactaa cctcaaaaca
aatgatgacc 300atgcacaaca ctaaaggacg aacctggtct cttatgctag
tatccttaat catttttatt 360gccacaacta acctcctcgg actcctgcct
cactcattta caccaaccac ccaactatct 420atgaacctag ccatggccat
ccccttatgg gcgggcacag tgattatggg ctttcgctct 480aagattaaaa
atgccctagc ccacttctta ccacaaggca cacctacacc ccttatcccc
540atgctagtta ttatcgaaac catcagccta ctcattcaac caatggccct
ggccgtacgc 600ctaaccgcta acattactgc aggccaccta ctcatgcacc
taattggaag cgccacccta 660gcaatgtcaa ccattaacct tccctctaca
cttatcatct tcacaattct aattctactg 720actatcctag aaatcgctgt
cgccttaatc caagcctacg ttttcacact tctagtaagc 780ctctacctgc
acgacaacac agcggccgcc cggtaagggc gaattcgata tcaagcttat
840cgataccgtc gacctcgagg attacaagga tgacgacgat aagtagggcc
cggtacctta 900attaattaag gtaccaggta agtgtaccca attcgcccta
tagtgagtcg tattacaatt 960cactcgatcg ttatgctgag tatgttaagc
tctttatgac tgtttttgta gtggtataga 1020gtactgcaga atacagtaag
ctgctctatt gtagcatttc ctgatgttgc ttagtcactt 1080atttcataaa
caacttaatg ttctgaataa tttcttacta aacattttgt tattgggcaa
1140gtgattgaaa atagtaaatg ctttgtgtga ttgacgcgt
1179231552DNAArtificial SequenceSynthetic COX10MTS-nND1
23gtcgacatgg ctgctagccc ccacactctg agcagccgcc tgctgaccgg ttgcgtgggc
60ggctctgtgt ggtatctgga gaggagaacc atgccaatgg caaatctgct gctcctcatc
120gtgccaatcc tgatcgccat ggccttcctc atgctgactg aaagaaaaat
tctgggatac 180atgcagctca ggaaggggcc taacgtggtg ggaccttatg
gactgctcca gccctttgct 240gatgctatga agctgttcac aaaagagccc
ctgaaaccag ccacctctac aatcaccctg 300tacattaccg ctcctaccct
ggctctgaca attgccctgc tgctgtggac ccctctccct 360atgccaaatc
ctctggtgaa cctgaatctg ggcctcctct ttatcctggc caccagcagc
420ctggccgtgt actccatcct gtggagcgga tgggcttcta acagcaatta
cgccctgatc 480ggtgccctga gggccgtggc ccagaccatt tcttacgagg
tgaccctcgc cattatcctg 540ctctcaaccc tgctgatgag cggctctttc
aacctctcaa ccctgattac aacccaggag 600cacctctggc tgctcctccc
cagctggcca ctggccatga tgtggtttat cagcaccctg 660gctgagacaa
accggacccc ctttgatctg gctgagggcg agtctgagct ggtctccgga
720ttcaatattg agtacgcagc agggccattc gctctgttct tcatggccga
gtatacaaat 780attattatga tgaacacact gactactact atcttcctgg
gtactacata cgatgctctg 840agtcccgaac tctacaccac ttacttcgtg
accaaaaccc tgctgctgac tagcctgttc 900ctgtggatca ggaccgccta
tccacgattc cgatacgacc agctgatgca tctgctgtgg 960aagaacttcc
tgccactcac cctggctctg ctcatgtggt acgtgagtat gccaatcact
1020atcagctcta tccctccaca gacctactcg aggaggatta caaggatgac
gacgataagt 1080agggcccggt accttaatta attaaggtac caggtaagtg
tacccaattc gccctatagt 1140gagtcgtatt acaattcact cgatcgccct
tcccaacagt tgcgcagcct gaatggcgaa 1200tggagatcca atttttaagt
gtataatgtg ttaaactact gattctaatt gtttgtgtat 1260tttagattca
cagtcccaag gctcatttca ggcccctcag tcctcacagt ctgttcatga
1320tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac
ctcccacacc 1380tccccctgaa cctgaaacat aaaatgaatg caattgttgt
tgttaacttg tttattgcag 1440cttataatgg ttacaaataa agcaatagca
tcacaaattt cacaaataaa gcattttttt 1500cactgcattc tagttgtggt
ttgtccaaac tcatcaatgt atcttaacgc gt 1552241973DNAArtificial
SequenceSynthetic COX10MTS-nND4 24gtcgacatgg ccgcctcacc ccacaccctg
agtagcaggc tgctgaccgg ctgtgtggga 60ggaagcgtgt ggtatctgga gcggagaacc
atgctgaagc tgatcgtgcc caccattatg 120ctgctgcctc tgacatggct
gtctaagaag cacatgatct ggattaacac aaccacccac 180agcctgatta
tctccatcat tcccctcctg ttcttcaacc agatcaacaa caacctgttc
240tcctgctcac ctacttttag cagcgatcca ctgacaaccc cactgctgat
gctgacaacc 300tggctcctcc ccctgacaat catggcttcc cagaggcacc
tgagcagcga gccactgtcc 360cgcaaaaagc tgtacctgtc catgctgatt
tctctccaga tctcactcat catgaccttc 420actgccaccg agctgattat
gttctatatc ttcttcgaga ctactctgat ccctacactc 480gccattatca
cccggtgggg caaccagcct gagagactga atgccgggac ttattttctg
540ttctacaccc tggtggggtc actgcccctg ctgattgccc tgatctacac
ccataacaca 600ctgggctctc tcaatatcct gctgctcaca ctgacagccc
aggagctgtc caattcttgg 660gctaacaatc tgatgtggct cgcatacact
atggccttca tggtgaagat gccactctat 720gggctccacc tctggctccc
taaggcccac gtcgaagccc caattgcagg gtccatggtg 780ctggcagctg
tgctcctgaa gctgggtggc tatgggatga tgcgcctgac cctgatcctg
840aatcctctca caaagcatat ggcttaccct tttctggtgc tgtccctgtg
gggaatgatt 900atgacaagct ctatttgcct gcgccagaca gacctgaaaa
gcctgattgc ctacagcagt 960atcagtcata tggccctggt ggtgaccgct
attctgattc agacaccatg gtcttttaca 1020ggggccgtca ttctgatgat
cgcccacgga ctgacctcat cactcctctt ctgtctggcc 1080aactcaaact
acgaaaggac acactcaaga attatgattc tgagccaggg actccagact
1140ctgctccccc tcatggcctt ctggtggctg ctcgcctctc tcgccaacct
ggccctccct 1200cccacaatca atctgctggg cgagctcagc gtgctggtga
ccacttttag ttggtccaac 1260atcacactgc tgctcaccgg actcaatatg
ctggtcaccg ccctgtacag tctgtacatg 1320ttcaccacaa cacagtgggg
tagcctcact catcacatta ataacatgaa gccttctttt 1380actagggaaa
atactctgat gtttatgcat ctctccccaa tcctcctcct gagtctgaac
1440cccgacatca tcaccggctt tagctctctc gaggaggatt acaaggatga
cgacgataag 1500tagggcccgg taccttaatt aattaaggta ccaggtaagt
gtacccaatt cgccctatag 1560tgagtcgtat tacaattcac tcgatcgccc
ttcccaacag ttgcgcagcc tgaatggcga 1620atggagatcc aatttttaag
tgtataatgt gttaaactac tgattctaat tgtttgtgta 1680ttttagattc
acagtcccaa ggctcatttc aggcccctca gtcctcacag tctgttcatg
1740atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa
cctcccacac 1800ctccccctga acctgaaaca taaaatgaat gcaattgttg
ttgttaactt gtttattgca 1860gcttataatg gttacaaata aagcaatagc
atcacaaatt tcacaaataa agcatttttt 1920tcactgcatt ctagttgtgg
tttgtccaaa ctcatcaatg tatcttaacg cgt 1973252595DNAArtificial
SequenceSynthetic COX10MTS-nND1-COX10 3'UTR 25gtcgacatgg ctgctagccc
ccacactctg agcagccgcc tgctgaccgg ttgcgtgggc 60ggctctgtgt ggtatctgga
gaggagaacc atgccaatgg caaatctgct gctcctcatc 120gtgccaatcc
tgatcgccat ggccttcctc atgctgactg aaagaaaaat tctgggatac
180atgcagctca ggaaggggcc taacgtggtg ggaccttatg gactgctcca
gccctttgct 240gatgctatga agctgttcac aaaagagccc ctgaaaccag
ccacctctac aatcaccctg 300tacattaccg ctcctaccct ggctctgaca
attgccctgc tgctgtggac ccctctccct 360atgccaaatc ctctggtgaa
cctgaatctg ggcctcctct ttatcctggc caccagcagc 420ctggccgtgt
actccatcct gtggagcgga tgggcttcta acagcaatta cgccctgatc
480ggtgccctga gggccgtggc ccagaccatt tcttacgagg tgaccctcgc
cattatcctg 540ctctcaaccc tgctgatgag cggctctttc aacctctcaa
ccctgattac aacccaggag 600cacctctggc tgctcctccc cagctggcca
ctggccatga tgtggtttat cagcaccctg 660gctgagacaa accggacccc
ctttgatctg gctgagggcg agtctgagct ggtctccgga 720ttcaatattg
agtacgcagc agggccattc gctctgttct tcatggccga gtatacaaat
780attattatga tgaacacact gactactact atcttcctgg gtactacata
cgatgctctg 840agtcccgaac tctacaccac ttacttcgtg accaaaaccc
tgctgctgac tagcctgttc 900ctgtggatca ggaccgccta tccacgattc
cgatacgacc agctgatgca tctgctgtgg 960aagaacttcc tgccactcac
cctggctctg ctcatgtggt acgtgagtat gccaatcact 1020atcagctcta
tccctccaca gacctactcg aggaggatta caaggatgac gacgataagt
1080agggcccggt accttaatta attaaggtac caggtaagtg tacccaattc
gccctatagt 1140gagtcgtatt acaattcact cgatcggagc actgggacgc
ccaccgcccc tttccctccg 1200ctgccaggcg agcatgttgt ggtaattctg
gaacacaaga agagaaattg ctgggtttag 1260aacaagatta taaacgaatt
cggtgctcag tgatcacttg acagtttttt ttttttttaa 1320atattaccca
aaatgctccc caaataagaa atgcatcagc tcagtcagtg aatacaaaaa
1380aggaattatt tttccctttg agggtcttta tacatctctc ctccaacccc
accctctatt 1440ctgtttcttc ctcctcacat gggggtacac atacacagct
tcctcttttg gttccatcct 1500taccaccaca ccacacgcac actccacatg
cccagcagag tggcacttgg tggccagaaa 1560gtgtgagcct catgatctgc
tgtctgtagt tctgtgagct caggtccctc aaaggcctcg 1620gagcaccccc
ttcctggtga ctgagccagg gcctgcattt ttggttttcc ccaccccaca
1680cattctcaac catagtcctt ctaacaatac caatagctag gacccggctg
ctgtgcactg 1740ggactgggga ttccacatgt ttgccttggg agtctcaagc
tggactgcca gcccctgtcc 1800tcccttcacc cccattgcgt atgagcattt
cagaactcca aggagtcaca ggcatcttta 1860tagttcacgt taacatatag
acactgttgg aagcagttcc ttctaaaagg gtagccctgg 1920acttaatacc
agccggatac ctctggcccc caccccatta ctgtacctct ggagtcacta
1980ctgtgggtcg ccactcctct gctacacagc acggcttttt caaggctgta
ttgagaaggg 2040aagttaggaa gaagggtgtg ctgggctaac cagcccacag
agctcacatt cctgtccctt 2100gggtgaaaaa tacatgtcca tcctgatatc
tcctgaattc agaaattagc ctccacatgt 2160gcaatggctt taagagccag
aagcagggtt ctgggaattt tgcaagttat cctgtggcca 2220ggtgtggtct
cggttaccaa atacggttac ctgcagcttt ttagtccttt gtgctcccac
2280gggtctgcag agtcccatct gcccaaaggt cttgaagctt gacaggatgt
tttcattact 2340cagtctccca gggcactgct ggtccgtagg gattcattgg
tcggggtggg agagttaaac 2400aacatttaaa cagagttctc tcaaaaatgt
ctaaagggat tgtaggtaga taacatccaa 2460tcactgtttg cacttatctg
aaatcttccc tcttggctgc ccccaggtat ttactgtgga 2520gaacattgca
taggaatgtc tgaaaaagct tctacaactt gttacagcct tcacatttgt
2580agaagcttta cgcgt 2595263016DNAArtificial SequenceSynthetic
COX10MTS-nND4-COX10 3'UTR 26gtcgacatgg ccgcctcacc ccacaccctg
agtagcaggc tgctgaccgg ctgtgtggga 60ggaagcgtgt ggtatctgga gcggagaacc
atgctgaagc tgatcgtgcc caccattatg 120ctgctgcctc tgacatggct
gtctaagaag cacatgatct ggattaacac aaccacccac
180agcctgatta tctccatcat tcccctcctg ttcttcaacc agatcaacaa
caacctgttc 240tcctgctcac ctacttttag cagcgatcca ctgacaaccc
cactgctgat gctgacaacc 300tggctcctcc ccctgacaat catggcttcc
cagaggcacc tgagcagcga gccactgtcc 360cgcaaaaagc tgtacctgtc
catgctgatt tctctccaga tctcactcat catgaccttc 420actgccaccg
agctgattat gttctatatc ttcttcgaga ctactctgat ccctacactc
480gccattatca cccggtgggg caaccagcct gagagactga atgccgggac
ttattttctg 540ttctacaccc tggtggggtc actgcccctg ctgattgccc
tgatctacac ccataacaca 600ctgggctctc tcaatatcct gctgctcaca
ctgacagccc aggagctgtc caattcttgg 660gctaacaatc tgatgtggct
cgcatacact atggccttca tggtgaagat gccactctat 720gggctccacc
tctggctccc taaggcccac gtcgaagccc caattgcagg gtccatggtg
780ctggcagctg tgctcctgaa gctgggtggc tatgggatga tgcgcctgac
cctgatcctg 840aatcctctca caaagcatat ggcttaccct tttctggtgc
tgtccctgtg gggaatgatt 900atgacaagct ctatttgcct gcgccagaca
gacctgaaaa gcctgattgc ctacagcagt 960atcagtcata tggccctggt
ggtgaccgct attctgattc agacaccatg gtcttttaca 1020ggggccgtca
ttctgatgat cgcccacgga ctgacctcat cactcctctt ctgtctggcc
1080aactcaaact acgaaaggac acactcaaga attatgattc tgagccaggg
actccagact 1140ctgctccccc tcatggcctt ctggtggctg ctcgcctctc
tcgccaacct ggccctccct 1200cccacaatca atctgctggg cgagctcagc
gtgctggtga ccacttttag ttggtccaac 1260atcacactgc tgctcaccgg
actcaatatg ctggtcaccg ccctgtacag tctgtacatg 1320ttcaccacaa
cacagtgggg tagcctcact catcacatta ataacatgaa gccttctttt
1380actagggaaa atactctgat gtttatgcat ctctccccaa tcctcctcct
gagtctgaac 1440cccgacatca tcaccggctt tagctctctc gaggaggatt
acaaggatga cgacgataag 1500tagggcccgg taccttaatt aattaaggta
ccaggtaagt gtacccaatt cgccctatag 1560tgagtcgtat tacaattcac
tcgatcggag cactgggacg cccaccgccc ctttccctcc 1620gctgccaggc
gagcatgttg tggtaattct ggaacacaag aagagaaatt gctgggttta
1680gaacaagatt ataaacgaat tcggtgctca gtgatcactt gacagttttt
ttttttttta 1740aatattaccc aaaatgctcc ccaaataaga aatgcatcag
ctcagtcagt gaatacaaaa 1800aaggaattat ttttcccttt gagggtcttt
atacatctct cctccaaccc caccctctat 1860tctgtttctt cctcctcaca
tgggggtaca catacacagc ttcctctttt ggttccatcc 1920ttaccaccac
accacacgca cactccacat gcccagcaga gtggcacttg gtggccagaa
1980agtgtgagcc tcatgatctg ctgtctgtag ttctgtgagc tcaggtccct
caaaggcctc 2040ggagcacccc cttcctggtg actgagccag ggcctgcatt
tttggttttc cccaccccac 2100acattctcaa ccatagtcct tctaacaata
ccaatagcta ggacccggct gctgtgcact 2160gggactgggg attccacatg
tttgccttgg gagtctcaag ctggactgcc agcccctgtc 2220ctcccttcac
ccccattgcg tatgagcatt tcagaactcc aaggagtcac aggcatcttt
2280atagttcacg ttaacatata gacactgttg gaagcagttc cttctaaaag
ggtagccctg 2340gacttaatac cagccggata cctctggccc ccaccccatt
actgtacctc tggagtcact 2400actgtgggtc gccactcctc tgctacacag
cacggctttt tcaaggctgt attgagaagg 2460gaagttagga agaagggtgt
gctgggctaa ccagcccaca gagctcacat tcctgtccct 2520tgggtgaaaa
atacatgtcc atcctgatat ctcctgaatt cagaaattag cctccacatg
2580tgcaatggct ttaagagcca gaagcagggt tctgggaatt ttgcaagtta
tcctgtggcc 2640aggtgtggtc tcggttacca aatacggtta cctgcagctt
tttagtcctt tgtgctccca 2700cgggtctgca gagtcccatc tgcccaaagg
tcttgaagct tgacaggatg ttttcattac 2760tcagtctccc agggcactgc
tggtccgtag ggattcattg gtcggggtgg gagagttaaa 2820caacatttaa
acagagttct ctcaaaaatg tctaaaggga ttgtaggtag ataacatcca
2880atcactgttt gcacttatct gaaatcttcc ctcttggctg cccccaggta
tttactgtgg 2940agaacattgc ataggaatgt ctgaaaaagc ttctacaact
tgttacagcc ttcacatttg 3000tagaagcttt acgcgt 301627696DNAArtificial
SequenceSynthetic nATP6 (recoded ATP6) 27atgaacgaaa atctgttcgc
ttcattcatt gcccccacaa tcctaggcct acccgccgca 60gtactgatca ttctatttcc
ccctctattg atccccacct ccaaatatct catcaacaac 120cgactaatca
ccacccaaca atggctaatc aaactaacct caaaacaaat gatgaccatg
180cacaacacta aaggacgaac ctggtctctt atgctagtat ccttaatcat
ttttattgcc 240acaactaacc tcctcggact cctgcctcac tcatttacac
caaccaccca actatctatg 300aacctagcca tggccatccc cttatgggcg
ggcacagtga ttatgggctt tcgctctaag 360attaaaaatg ccctagccca
cttcttacca caaggcacac ctacacccct tatccccatg 420ctagttatta
tcgaaaccat cagcctactc attcaaccaa tggccctggc cgtacgccta
480accgctaaca ttactgcagg ccacctactc atgcacctaa ttggaagcgc
caccctagca 540atgtcaacca ttaaccttcc ctctacactt atcatcttca
caattctaat tctactgact 600atcctagaaa tcgctgtcgc cttaatccaa
gcctacgttt tcacacttct agtaagcctc 660tacctgcacg acaacacagc
ggccgcccgg aagggc 69628956DNAArtificial SequenceSynthetic nND1
(recoded ND1) 28atgccaatgg caaatctgct gctcctcatc gtgccaatcc
tgatcgccat ggccttcctc 60atgctgactg aaagaaaaat tctgggatac atgcagctca
ggaaggggcc taacgtggtg 120ggaccttatg gactgctcca gccctttgct
gatgctatga agctgttcac aaaagagccc 180ctgaaaccag ccacctctac
aatcaccctg tacattaccg ctcctaccct ggctctgaca 240attgccctgc
tgctgtggac ccctctccct atgccaaatc ctctggtgaa cctgaatctg
300ggcctcctct ttatcctggc caccagcagc ctggccgtgt actccatcct
gtggagcgga 360tgggcttcta acagcaatta cgccctgatc ggtgccctga
gggccgtggc ccagaccatt 420tcttacgagg tgaccctcgc cattatcctg
ctctcaaccc tgctgatgag cggctctttc 480aacctctcaa ccctgattac
aacccaggag cacctctggc tgctcctccc cagctggcca 540ctggccatga
tgtggtttat cagcaccctg gctgagacaa accggacccc ctttgatctg
600gctgagggcg agtctgagct ggtctccgga ttcaatattg agtacgcagc
agggccattc 660gctctgttct tcatggccga gtatacaaat attattatga
tgaacacact gactactact 720atcttcctgg gtactacata cgatgctctg
agtcccgaac tctacaccac ttacttcgtg 780accaaaaccc tgctgctgac
tagcctgttc ctgtggatca ggaccgccta tccacgattc 840cgatacgacc
agctgatgca tctgctgtgg aagaacttcc tgccactcac cctggctctg
900ctcatgtggt acgtgagtat gccaatcact atcagctcta tccctccaca gaccta
956291377DNAArtificial SequenceSynthetic nND4 (recoded ND4)
29atgctgaagc tgatcgtgcc caccattatg ctgctgcctc tgacatggct gtctaagaag
60cacatgatct ggattaacac aaccacccac agcctgatta tctccatcat tcccctcctg
120ttcttcaacc agatcaacaa caacctgttc tcctgctcac ctacttttag
cagcgatcca 180ctgacaaccc cactgctgat gctgacaacc tggctcctcc
ccctgacaat catggcttcc 240cagaggcacc tgagcagcga gccactgtcc
cgcaaaaagc tgtacctgtc catgctgatt 300tctctccaga tctcactcat
catgaccttc actgccaccg agctgattat gttctatatc 360ttcttcgaga
ctactctgat ccctacactc gccattatca cccggtgggg caaccagcct
420gagagactga atgccgggac ttattttctg ttctacaccc tggtggggtc
actgcccctg 480ctgattgccc tgatctacac ccataacaca ctgggctctc
tcaatatcct gctgctcaca 540ctgacagccc aggagctgtc caattcttgg
gctaacaatc tgatgtggct cgcatacact 600atggccttca tggtgaagat
gccactctat gggctccacc tctggctccc taaggcccac 660gtcgaagccc
caattgcagg gtccatggtg ctggcagctg tgctcctgaa gctgggtggc
720tatgggatga tgcgcctgac cctgatcctg aatcctctca caaagcatat
ggcttaccct 780tttctggtgc tgtccctgtg gggaatgatt atgacaagct
ctatttgcct gcgccagaca 840gacctgaaaa gcctgattgc ctacagcagt
atcagtcata tggccctggt ggtgaccgct 900attctgattc agacaccatg
gtcttttaca ggggccgtca ttctgatgat cgcccacgga 960ctgacctcat
cactcctctt ctgtctggcc aactcaaact acgaaaggac acactcaaga
1020attatgattc tgagccaggg actccagact ctgctccccc tcatggcctt
ctggtggctg 1080ctcgcctctc tcgccaacct ggccctccct cccacaatca
atctgctggg cgagctcagc 1140gtgctggtga ccacttttag ttggtccaac
atcacactgc tgctcaccgg actcaatatg 1200ctggtcaccg ccctgtacag
tctgtacatg ttcaccacaa cacagtgggg tagcctcact 1260catcacatta
ataacatgaa gccttctttt actagggaaa atactctgat gtttatgcat
1320ctctccccaa tcctcctcct gagtctgaac cccgacatca tcaccggctt tagctct
13773084DNAHomo sapiens 30atggccgcat ctccgcacac tctctcctca
cgcctcctga caggttgcgt aggaggctct 60gtctggtatc ttgaagtcga ccgc
8431102DNAHomo sapiens 31atgttgagcc gggcagtgtg cggcaccagc
aggcagctgg ctccggtttt ggggtatctg 60ggctccaggc agaagcacag cctccccgac
gcggtcgacc gc 1023230PRTHomo sapiens 32Met Ala Pro Tyr Ser Leu Leu
Val Thr Arg Leu Gln Lys Ala Leu Gly 1 5 10 15 Val Arg Gln Tyr His
Val Ala Ser Val Leu Cys Gln Arg Ala 20 25 30 33382DNAHomo sapiens
33gggcagtgcc tccccgcccc gccgctggcg tcaagttcag ctccacgtgt gccatcagtg
60gatccgatcc gtccagccat ggcttcctat tccaagatgg tgtgaccaga catgcttcct
120gctccccgct tagcccacgg agtgactgtg gttgtggtgg gggggttctt
aaaataactt 180tttagccccc gtcttcctat tttgagtttg gttcagatct
taagcagctc catgcaactg 240tatttatttt tgatgacaag actcccatct
aaagtttttc tcctgcctga tcatttcatt 300ggtggctgaa ggattctaga
gaaccttttg ttcttgcaag gaaaacaaga atccaaaacc 360agaaaaaaaa
aaaaaaaaaa aa 3823420PRTHomo sapiens 34Met Leu Ser Arg Ala Val Cys
Gly Thr Ser Arg Gln Leu Ala Pro Ala 1 5 10 15 Leu Gly Tyr Leu 20
35214DNAHomo sapiens 35accacgatcg ttatgctgat cataccctaa tgatcccagc
aagataacgt cctgtcttct 60aagatgtgca tcaagcctgg tacatactga aaaccctata
aggtcctgga taatttttgt 120ttgattattc attgaagaaa catttatttt
ccaattgtgt gaagtttttg actgttaata 180aaagaatctg tcaaccatca
aaaaaaaaaa aaaa 2143663PRTHomo sapiens 36Met Leu Gly Phe Val Gly
Arg Val Ala Ala Ala Pro Ala Ser Gly Ala 1 5 10 15 Leu Arg Arg Leu
Thr Pro Ser Ala Ser Leu Pro Pro Ala Gln Leu Leu 20 25 30 Leu Arg
Ala Ala Pro Thr Ala Val His Pro Val Arg Asp Tyr Ala Ala 35 40 45
Gln Thr Ser Pro Ser Pro Lys Ala Gly Ala Ala Thr Gly Arg Ile 50 55
60 37162DNAHomo sapiens 37ggggtctttg tcctctgtac tgtctctctc
cttgccccta acccaaaaag cttcattttt 60ctgtgtaggc tgcacaagag ccttgattga
agatatattc tttctgaaca gtatttaagg 120tttccaataa aatgtacacc
cctcagaaaa aaaaaaaaaa aa 1623836PRTHomo sapiens 38Met Leu Ser Val
Ala Ala Arg Ser Gly Pro Phe Ala Pro Val Leu Ser 1 5 10 15 Ala Thr
Ser Arg Gly Val Ala Gly Ala Leu Arg Pro Leu Val Gln Ala 20 25 30
Thr Val Pro Ala 35 39233DNAHomo sapiens 39gagacttgga ctcaagtcat
aggcttcttt cagtctttat gtcacctcag gagacttatt 60tgagaggaag ccttctgtac
ttgaagttga tttgaaatat gtaagaattg atgatgtatt 120tgcaaacatt
aatgtgaaat aaattgaatt taatgttgaa tactttcagg cattcactta
180ataaagacac tgttaagcac tgttatgctc agtcaaaaaa aaaaaaaaaa aaa
2334020PRTHomo sapiens 40Met Leu Ala Thr Arg Arg Leu Leu Gly Trp
Ser Leu Pro Ala Arg Val 1 5 10 15 Ser Val Arg Phe 20 41102DNAHomo
sapiens 41cccaccaccc tggcctgctg tcctgcgtct atccatgtgg aatgctggac
aataaagcga 60gtgctgccca ccctccaaaa aaaaaaaaaa aaaaaaaaaa aa
1024243PRTHomo sapiens 42Met Phe Phe Ser Ala Ala Leu Arg Ala Arg
Ala Ala Gly Leu Thr Ala 1 5 10 15 His Trp Gly Arg His Val Arg Asn
Leu His Lys Thr Ala Met Gln Asn 20 25 30 Gly Ala Gly Gly Ala Leu
Phe Val His Arg Asp 35 40 4397DNAHomo sapiens 43tttatattga
actgtaaata tgtcactaga gaaataaaat atggacttcc aatctacgta 60aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaa 974424PRTHomo sapiens 44Met Leu Arg
Ala Ala Ala Arg Phe Gly Pro Arg Leu Gly Arg Arg Leu 1 5 10 15 Leu
Ser Ala Ala Ala Thr Gln Ala 20 45450DNAHomo sapiens 45gaatcatgca
agcttcctcc ctcagccatt gatggaaagt tcagcaagat cagcaacaaa 60accaagaaaa
atgatccttg cgtgctgaat atctgaaaag agaaattttt cctacaaaat
120ctcttgggtc aagaaagttc tagaatttga attgataaac atggtgggtt
ggctgagggt 180aagagtatat gaggaacctt ttaaacgaca acaatactgc
tagctttcag gatgattttt 240aaaaaataga ttcaaatgtg ttatcctctc
tctgaaacgc ttcctataac tcgagtttat 300aggggaagaa aaagctattg
tttacaatta tatccccatt aaggcaactg ctacaccctg 360ctttgtattc
tgggctaaga ttcattaaaa actagctgct cttaaaaaaa aaaaaaaaaa
420aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4504620PRTHomo sapiens 46Met
Ala Ala Ser Pro His Thr Leu Ser Ser Arg Leu Leu Thr Gly Cys 1 5 10
15 Val Gly Gly Ser 20 471424DNAHomo sapiens 47gagcactggg acgcccaccg
cccctttccc tccgctgcca ggcgagcatg ttgtggtaat 60tctggaacac aagaagagaa
attgctgggt ttagaacaag attataaacg aattcggtgc 120ccagtgatca
cttgacagtt tttttttttt ttaaatatta cccaaaatgc tccccaaata
180agaaatgcat cagctcagtc agtgaataca aaaaaggaat tatttttccc
tttgagggtc 240tttatacatc tctcctccaa ccccaccctc tattctgttt
cttcctcctc acatgggggt 300acacatacac agcttcctct tttggttcca
tccttaccac cacaccacac gcacactcca 360catgcccagc agagtggcac
ttggtggcca gaaagtgtga gcctcatgat ctgctgtctg 420tagttctgtg
agctcaggtc cctcaaaggc ctcggagcac ccccttcctg gtgactgagc
480cagggcctgc atttttggtt ttccccaccc cacacattct caaccatagt
ccttctaaca 540ataccaatag ctaggacccg gctgctgtgc actgggactg
gggattccac atgtttgcct 600tgggagtctc aagctggact gccagcccct
gtcctccctt cacccccatt gcgtatgagc 660atttcagaac tccaaggagt
cacaggcatc tttatagttc acgttaacat atagacactg 720ttggaagcag
ttccttctaa aagggtagcc ctggacttaa taccagccgg atacctctgg
780cccccacccc attactgtac ctctggagtc actactgtgg gtcgccactc
ctctgctaca 840cagcacggct ttttcaaggc tgtattgaga agggaagtta
ggaagaaggg tgtgctgggc 900taaccagccc acagagctca cattcctgtc
ccttgggtga aaaatacatg tccatcctga 960tatctcctga attcagaaat
tagcctccac atgtgcaatg gctttaagag ccagaagcag 1020ggttctggga
attttgcaag ttatcctgtg gccaggtgtg gtctcggtta ccaaatacgg
1080ttacctgcag ctttttagtc ctttgtgctc ccacgggtct gcagagtccc
atctgcccaa 1140aggtcttgaa gcttgacagg atgttttcat tactcagtct
cccagggcac tgctggtccg 1200tagggattca ttggtcgggg tgggagagtt
aaacaacatt taaacagagt tctctcaaaa 1260atgtctaaag ggattgtagg
tagataacat ccaatcactg tttgcactta tctgaaatct 1320tccctcttgg
ctgcccccag gtatttactg tggagaacat tgcataggaa tgtctggaaa
1380aagctcctac aacttgttac agccttcaca tttgtagaag cttt
142448780PRTHomo sapiens 48Met Ala Pro Tyr Ser Leu Leu Val Thr Arg
Leu Gln Lys Ala Leu Gly 1 5 10 15 Val Arg Gln Tyr His Val Ala Ser
Val Leu Cys Gln Arg Ala Lys Val 20 25 30 Ala Met Ser His Phe Glu
Pro Asn Glu Tyr Ile His Tyr Asp Leu Leu 35 40 45 Glu Lys Asn Ile
Asn Ile Val Arg Lys Arg Leu Asn Arg Pro Leu Thr 50 55 60 Leu Ser
Glu Lys Ile Val Tyr Gly His Leu Asp Asp Pro Ala Ser Gln 65 70 75 80
Glu Ile Glu Arg Gly Lys Ser Tyr Leu Arg Leu Arg Pro Asp Arg Val 85
90 95 Ala Met Gln Asp Ala Thr Ala Gln Met Ala Met Leu Gln Phe Ile
Ser 100 105 110 Ser Gly Leu Ser Lys Val Ala Val Pro Ser Thr Ile His
Cys Asp His 115 120 125 Leu Ile Glu Ala Gln Val Gly Gly Glu Lys Asp
Leu Arg Arg Ala Lys 130 135 140 Asp Ile Asn Gln Glu Val Tyr Asn Phe
Leu Ala Thr Ala Gly Ala Lys 145 150 155 160 Tyr Gly Val Gly Phe Trp
Lys Pro Gly Ser Gly Ile Ile His Gln Ile 165 170 175 Ile Leu Glu Asn
Tyr Ala Tyr Pro Gly Val Leu Leu Ile Gly Thr Asp 180 185 190 Ser His
Thr Pro Asn Gly Gly Gly Leu Gly Gly Ile Cys Ile Gly Val 195 200 205
Gly Gly Ala Asp Ala Val Asp Val Met Ala Gly Ile Pro Trp Glu Leu 210
215 220 Lys Cys Pro Lys Val Ile Gly Val Lys Leu Thr Gly Ser Leu Ser
Gly 225 230 235 240 Trp Ser Ser Pro Lys Asp Val Ile Leu Lys Val Ala
Gly Ile Leu Thr 245 250 255 Val Lys Gly Gly Thr Gly Ala Ile Val Glu
Tyr His Gly Pro Gly Val 260 265 270 Asp Ser Ile Ser Cys Thr Gly Met
Ala Thr Ile Cys Asn Met Gly Ala 275 280 285 Glu Ile Gly Ala Thr Thr
Ser Val Phe Pro Tyr Asn His Arg Met Lys 290 295 300 Lys Tyr Leu Ser
Lys Thr Gly Arg Glu Asp Ile Ala Asn Leu Ala Asp 305 310 315 320 Glu
Phe Lys Asp His Leu Val Pro Asp Pro Gly Cys His Tyr Asp Gln 325 330
335 Leu Ile Glu Ile Asn Leu Ser Glu Leu Lys Pro His Ile Asn Gly Pro
340 345 350 Phe Thr Pro Asp Leu Ala His Pro Val Ala Glu Val Gly Lys
Val Ala 355 360 365 Glu Lys Glu Gly Trp Pro Leu Asp Ile Arg Val Gly
Leu Ile Gly Ser 370 375 380 Cys Thr Asn Ser Ser Tyr Glu Asp Met Gly
Arg Ser Ala Ala Val Ala 385 390 395 400 Lys Gln Ala Leu Ala His Gly
Leu Lys Cys Lys Ser Gln Phe Thr Ile 405 410 415 Thr Pro Gly Ser Glu
Gln Ile Arg Ala Thr Ile Glu Arg Asp Gly Tyr 420 425 430 Ala Gln Ile
Leu Arg Asp Leu Gly Gly Ile Val Leu Ala Asn Ala Cys 435 440 445 Gly
Pro Cys Ile Gly Gln Trp Asp Arg Lys Asp Ile Lys Lys Gly Glu 450 455
460 Lys Asn Thr Ile Val Thr Ser Tyr Asn Arg Asn Phe Thr Gly Arg Asn
465 470 475 480 Asp Ala Asn Pro Glu Thr His Ala Phe Val Thr Ser Pro
Glu Ile Val 485 490 495 Thr Ala Leu Ala Ile Ala Gly Thr Leu Lys Phe
Asn Pro Glu Thr Asp 500 505 510 Tyr Leu Thr Gly Thr Asp Gly Lys Lys
Phe Arg Leu Glu Ala Pro Asp
515 520 525 Ala Asp Glu Leu Pro Lys Gly Glu Phe Asp Pro Gly Gln Asp
Thr Tyr 530 535 540 Gln His Pro Pro Lys Asp Ser Ser Gly Gln His Val
Asp Val Ser Pro 545 550 555 560 Thr Ser Gln Arg Leu Gln Leu Leu Glu
Pro Phe Asp Lys Trp Asp Gly 565 570 575 Lys Asp Leu Glu Asp Leu Gln
Ile Leu Ile Lys Val Lys Gly Lys Cys 580 585 590 Thr Thr Asp His Ile
Ser Ala Ala Gly Pro Trp Leu Lys Phe Arg Gly 595 600 605 His Leu Asp
Asn Ile Ser Asn Asn Leu Leu Ile Gly Ala Ile Asn Ile 610 615 620 Glu
Asn Gly Lys Ala Asn Ser Val Arg Asn Ala Val Thr Gln Glu Phe 625 630
635 640 Gly Pro Val Pro Asp Thr Ala Arg Tyr Tyr Lys Lys His Gly Ile
Arg 645 650 655 Trp Val Val Ile Gly Asp Glu Asn Tyr Gly Glu Gly Ser
Ser Arg Glu 660 665 670 His Ala Ala Leu Glu Pro Arg His Leu Gly Gly
Arg Ala Ile Ile Thr 675 680 685 Lys Ser Phe Ala Arg Ile His Glu Thr
Asn Leu Lys Lys Gln Gly Leu 690 695 700 Leu Pro Leu Thr Phe Ala Asp
Pro Ala Asp Tyr Asn Lys Ile His Pro 705 710 715 720 Val Asp Lys Leu
Thr Ile Gln Gly Leu Lys Asp Phe Thr Pro Gly Lys 725 730 735 Pro Leu
Lys Cys Ile Ile Lys His Pro Asn Gly Thr Gln Glu Thr Ile 740 745 750
Leu Leu Asn His Thr Phe Asn Glu Thr Gln Ile Glu Trp Phe Arg Ala 755
760 765 Gly Ser Ala Leu Asn Arg Met Lys Glu Leu Gln Gln 770 775 780
49222PRTHomo sapiens 49Met Leu Ser Arg Ala Val Cys Gly Thr Ser Arg
Gln Leu Ala Pro Ala 1 5 10 15 Leu Gly Tyr Leu Gly Ser Arg Gln Lys
His Ser Leu Pro Asp Leu Pro 20 25 30 Tyr Asp Tyr Gly Ala Leu Glu
Pro His Ile Asn Ala Gln Ile Met Gln 35 40 45 Leu His His Ser Lys
His His Ala Ala Tyr Val Asn Asn Leu Asn Val 50 55 60 Thr Glu Glu
Lys Tyr Gln Glu Ala Leu Ala Lys Gly Asp Val Thr Ala 65 70 75 80 Gln
Ile Ala Leu Gln Pro Ala Leu Lys Phe Asn Gly Gly Gly His Ile 85 90
95 Asn His Ser Ile Phe Trp Thr Asn Leu Ser Pro Asn Gly Gly Gly Glu
100 105 110 Pro Lys Gly Glu Leu Leu Glu Ala Ile Lys Arg Asp Phe Gly
Ser Phe 115 120 125 Asp Lys Phe Lys Glu Lys Leu Thr Ala Ala Ser Val
Gly Val Gln Gly 130 135 140 Ser Gly Trp Gly Trp Leu Gly Phe Asn Lys
Glu Arg Gly His Leu Gln 145 150 155 160 Ile Ala Ala Cys Pro Asn Gln
Asp Pro Leu Gln Gly Thr Thr Gly Leu 165 170 175 Ile Pro Leu Leu Gly
Ile Asp Val Trp Glu His Ala Tyr Tyr Leu Gln 180 185 190 Tyr Lys Asn
Val Arg Pro Asp Tyr Leu Lys Ala Ile Trp Asn Val Ile 195 200 205 Asn
Trp Glu Asn Val Thr Glu Arg Tyr Met Ala Cys Lys Lys 210 215 220
50529PRTHomo sapiens 50Met Leu Gly Phe Val Gly Arg Val Ala Ala Ala
Pro Ala Ser Gly Ala 1 5 10 15 Leu Arg Arg Leu Thr Pro Ser Ala Ser
Leu Pro Pro Ala Gln Leu Leu 20 25 30 Leu Arg Ala Ala Pro Thr Ala
Val His Pro Val Arg Asp Tyr Ala Ala 35 40 45 Gln Thr Ser Pro Ser
Pro Lys Ala Gly Ala Ala Thr Gly Arg Ile Val 50 55 60 Ala Val Ile
Gly Ala Val Val Asp Val Gln Phe Asp Glu Gly Leu Pro 65 70 75 80 Pro
Ile Leu Asn Ala Leu Glu Val Gln Gly Arg Glu Thr Arg Leu Val 85 90
95 Leu Glu Val Ala Gln His Leu Gly Glu Ser Thr Val Arg Thr Ile Ala
100 105 110 Met Asp Gly Thr Glu Gly Leu Val Arg Gly Gln Lys Val Leu
Asp Ser 115 120 125 Gly Ala Pro Ile Lys Ile Pro Val Gly Pro Glu Thr
Leu Gly Arg Ile 130 135 140 Met Asn Val Ile Gly Glu Pro Ile Asp Glu
Arg Gly Pro Ile Lys Thr 145 150 155 160 Lys Gln Phe Ala Pro Ile His
Ala Glu Ala Pro Glu Phe Met Glu Met 165 170 175 Ser Val Glu Gln Glu
Ile Leu Val Thr Gly Ile Lys Val Val Asp Leu 180 185 190 Leu Ala Pro
Tyr Ala Lys Gly Gly Lys Ile Gly Leu Phe Gly Gly Ala 195 200 205 Gly
Val Gly Lys Thr Val Leu Ile Met Glu Leu Ile Asn Asn Val Ala 210 215
220 Lys Ala His Gly Gly Tyr Ser Val Phe Ala Gly Val Gly Glu Arg Thr
225 230 235 240 Arg Glu Gly Asn Asp Leu Tyr His Glu Met Ile Glu Ser
Gly Val Ile 245 250 255 Asn Leu Lys Asp Ala Thr Ser Lys Val Ala Leu
Val Tyr Gly Gln Met 260 265 270 Asn Glu Pro Pro Gly Ala Arg Ala Arg
Val Ala Leu Thr Gly Leu Thr 275 280 285 Val Ala Glu Tyr Phe Arg Asp
Gln Glu Gly Gln Asp Val Leu Leu Phe 290 295 300 Ile Asp Asn Ile Phe
Arg Phe Thr Gln Ala Gly Ser Glu Val Ser Ala 305 310 315 320 Leu Leu
Gly Arg Ile Pro Ser Ala Val Gly Tyr Gln Pro Thr Leu Ala 325 330 335
Thr Asp Met Gly Thr Met Gln Glu Arg Ile Thr Thr Thr Lys Lys Gly 340
345 350 Ser Ile Thr Ser Val Gln Ala Ile Tyr Val Pro Ala Asp Asp Leu
Thr 355 360 365 Asp Pro Ala Pro Ala Thr Thr Phe Ala His Leu Asp Ala
Thr Thr Val 370 375 380 Leu Ser Arg Ala Ile Ala Glu Leu Gly Ile Tyr
Pro Ala Val Asp Pro 385 390 395 400 Leu Asp Ser Thr Ser Arg Ile Met
Asp Pro Asn Ile Val Gly Ser Glu 405 410 415 His Tyr Asp Val Ala Arg
Gly Val Gln Lys Ile Leu Gln Asp Tyr Lys 420 425 430 Ser Leu Gln Asp
Ile Ile Ala Ile Leu Gly Met Asp Glu Leu Ser Glu 435 440 445 Glu Asp
Lys Leu Thr Val Ser Arg Ala Arg Lys Ile Gln Arg Phe Leu 450 455 460
Ser Gln Pro Phe Gln Val Ala Glu Val Phe Thr Gly His Met Gly Lys 465
470 475 480 Leu Val Pro Leu Lys Glu Thr Ile Lys Gly Phe Gln Gln Ile
Leu Ala 485 490 495 Gly Glu Tyr Asp His Leu Pro Glu Gln Ala Phe Tyr
Met Val Gly Pro 500 505 510 Ile Glu Glu Ala Val Ala Lys Ala Asp Lys
Leu Ala Glu Glu His Ser 515 520 525 Ser 51274PRTHomo sapiens 51Met
Leu Ser Val Ala Ala Arg Ser Gly Pro Phe Ala Pro Val Leu Ser 1 5 10
15 Ala Thr Ser Arg Gly Val Ala Gly Ala Leu Arg Pro Leu Val Gln Ala
20 25 30 Thr Val Pro Ala Thr Pro Glu Gln Pro Val Leu Asp Leu Lys
Arg Pro 35 40 45 Phe Leu Ser Arg Glu Ser Leu Ser Gly Gln Ala Val
Arg Arg Pro Leu 50 55 60 Val Ala Ser Val Gly Leu Asn Val Pro Ala
Ser Val Cys Tyr Ser His 65 70 75 80 Thr Asp Ile Lys Val Pro Asp Phe
Ser Glu Tyr Arg Arg Leu Glu Val 85 90 95 Leu Asp Ser Thr Lys Ser
Ser Arg Glu Ser Ser Glu Ala Arg Lys Gly 100 105 110 Phe Ser Tyr Leu
Val Thr Gly Val Thr Thr Val Gly Val Ala Tyr Ala 115 120 125 Ala Lys
Asn Ala Val Thr Gln Phe Val Ser Ser Met Ser Ala Ser Ala 130 135 140
Asp Val Leu Ala Leu Ala Lys Ile Glu Ile Lys Leu Ser Asp Ile Pro 145
150 155 160 Glu Gly Lys Asn Met Ala Phe Lys Trp Arg Gly Lys Pro Leu
Phe Val 165 170 175 Arg His Arg Thr Gln Lys Glu Ile Glu Gln Glu Ala
Ala Val Glu Leu 180 185 190 Ser Gln Leu Arg Asp Pro Gln His Asp Leu
Asp Arg Val Lys Lys Pro 195 200 205 Glu Trp Val Ile Leu Ile Gly Val
Cys Thr His Leu Gly Cys Val Pro 210 215 220 Ile Ala Asn Ala Gly Asp
Phe Gly Gly Tyr Tyr Cys Pro Cys His Gly 225 230 235 240 Ser His Tyr
Asp Ala Ser Gly Arg Ile Arg Leu Gly Pro Ala Pro Leu 245 250 255 Asn
Leu Glu Val Pro Thr Tyr Glu Phe Thr Ser Asp Asp Met Val Ile 260 265
270 Val Gly 52464PRTHomo sapiens 52Met Leu Ala Thr Arg Arg Leu Leu
Gly Trp Ser Leu Pro Ala Arg Val 1 5 10 15 Ser Val Arg Phe Ser Gly
Asp Thr Thr Ala Pro Lys Lys Thr Ser Phe 20 25 30 Gly Ser Leu Lys
Asp Glu Asp Arg Ile Phe Thr Asn Leu Tyr Gly Arg 35 40 45 His Asp
Trp Arg Leu Lys Gly Ser Leu Ser Arg Gly Asp Trp Tyr Lys 50 55 60
Thr Lys Glu Ile Leu Leu Lys Gly Pro Asp Trp Ile Leu Gly Glu Ile 65
70 75 80 Lys Thr Ser Gly Leu Arg Gly Arg Gly Gly Ala Gly Phe Pro
Thr Gly 85 90 95 Leu Lys Trp Ser Phe Met Asn Lys Pro Ser Asp Gly
Arg Pro Lys Tyr 100 105 110 Leu Val Val Asn Ala Asp Glu Gly Glu Pro
Gly Thr Cys Lys Asp Arg 115 120 125 Glu Ile Leu Arg His Asp Pro His
Lys Leu Leu Glu Gly Cys Leu Val 130 135 140 Gly Gly Arg Ala Met Gly
Ala Arg Ala Ala Tyr Ile Tyr Ile Arg Gly 145 150 155 160 Glu Phe Tyr
Asn Glu Ala Ser Asn Leu Gln Val Ala Ile Arg Glu Ala 165 170 175 Tyr
Glu Ala Gly Leu Ile Gly Lys Asn Ala Cys Gly Ser Gly Tyr Asp 180 185
190 Phe Asp Val Phe Val Val Arg Gly Ala Gly Ala Tyr Ile Cys Gly Glu
195 200 205 Glu Thr Ala Leu Ile Glu Ser Ile Glu Gly Lys Gln Gly Lys
Pro Arg 210 215 220 Leu Lys Pro Pro Phe Pro Ala Asp Val Gly Val Phe
Gly Cys Pro Thr 225 230 235 240 Thr Val Ala Asn Val Glu Thr Val Ala
Val Ser Pro Thr Ile Cys Arg 245 250 255 Arg Gly Gly Thr Trp Phe Ala
Gly Phe Gly Arg Glu Arg Asn Ser Gly 260 265 270 Thr Lys Leu Phe Asn
Ile Ser Gly His Val Asn His Pro Cys Thr Val 275 280 285 Glu Glu Glu
Met Ser Val Pro Leu Lys Glu Leu Ile Glu Lys His Ala 290 295 300 Gly
Gly Val Thr Gly Gly Trp Asp Asn Leu Leu Ala Val Ile Pro Gly 305 310
315 320 Gly Ser Ser Thr Pro Leu Ile Pro Lys Ser Val Cys Glu Thr Val
Leu 325 330 335 Met Asp Phe Asp Ala Leu Val Gln Ala Gln Thr Gly Leu
Gly Thr Ala 340 345 350 Ala Val Ile Val Met Asp Arg Ser Thr Asp Ile
Val Lys Ala Ile Ala 355 360 365 Arg Leu Ile Glu Phe Tyr Lys His Glu
Ser Cys Gly Gln Cys Thr Pro 370 375 380 Cys Arg Glu Gly Val Asp Trp
Met Asn Lys Val Met Ala Arg Phe Val 385 390 395 400 Arg Gly Asp Ala
Arg Pro Ala Glu Ile Asp Ser Leu Trp Glu Ile Ser 405 410 415 Lys Gln
Ile Glu Gly His Thr Ile Cys Ala Leu Gly Asp Gly Ala Ala 420 425 430
Trp Pro Val Gln Gly Leu Ile Arg His Phe Arg Pro Glu Leu Glu Glu 435
440 445 Arg Met Gln Arg Phe Ala Gln Gln His Gln Ala Arg Gln Ala Ala
Ser 450 455 460 53249PRTHomo sapiens 53Met Phe Phe Ser Ala Ala Leu
Arg Ala Arg Ala Ala Gly Leu Thr Ala 1 5 10 15 His Trp Gly Arg His
Val Arg Asn Leu His Lys Thr Ala Met Gln Asn 20 25 30 Gly Ala Gly
Gly Ala Leu Phe Val His Arg Asp Thr Pro Glu Asn Asn 35 40 45 Pro
Asp Thr Pro Phe Asp Phe Thr Pro Glu Asn Tyr Lys Arg Ile Glu 50 55
60 Ala Ile Val Lys Asn Tyr Pro Glu Gly His Lys Ala Ala Ala Val Leu
65 70 75 80 Pro Val Leu Asp Leu Ala Gln Arg Gln Asn Gly Trp Leu Pro
Ile Ser 85 90 95 Ala Met Asn Lys Val Ala Glu Val Leu Gln Val Pro
Pro Met Arg Val 100 105 110 Tyr Glu Val Ala Thr Phe Tyr Thr Met Tyr
Asn Arg Lys Pro Val Gly 115 120 125 Lys Tyr His Ile Gln Val Cys Thr
Thr Thr Pro Cys Met Leu Arg Asn 130 135 140 Ser Asp Ser Ile Leu Glu
Ala Ile Gln Lys Lys Leu Gly Ile Lys Val 145 150 155 160 Gly Glu Thr
Thr Pro Asp Lys Leu Phe Thr Leu Ile Glu Val Glu Cys 165 170 175 Leu
Gly Ala Cys Val Asn Ala Pro Met Val Gln Ile Asn Asp Asn Tyr 180 185
190 Tyr Glu Asp Leu Thr Ala Lys Asp Ile Glu Glu Ile Ile Asp Glu Leu
195 200 205 Lys Ala Gly Lys Ile Pro Lys Pro Gly Pro Arg Ser Gly Arg
Phe Ser 210 215 220 Cys Glu Pro Ala Gly Gly Leu Thr Ser Leu Thr Glu
Pro Pro Lys Gly 225 230 235 240 Pro Gly Phe Gly Val Gln Ala Gly Leu
245 54517PRTHomo sapiens 54Met Leu Arg Ala Ala Ala Arg Phe Gly Pro
Arg Leu Gly Arg Arg Leu 1 5 10 15 Leu Ser Ala Ala Ala Thr Gln Ala
Val Pro Ala Pro Asn Gln Gln Pro 20 25 30 Glu Val Phe Cys Asn Gln
Ile Phe Ile Asn Asn Glu Trp His Asp Ala 35 40 45 Val Ser Arg Lys
Thr Phe Pro Thr Val Asn Pro Ser Thr Gly Glu Val 50 55 60 Ile Cys
Gln Val Ala Glu Gly Asp Lys Glu Asp Val Asp Lys Ala Val 65 70 75 80
Lys Ala Ala Arg Ala Ala Phe Gln Leu Gly Ser Pro Trp Arg Arg Met 85
90 95 Asp Ala Ser His Arg Gly Arg Leu Leu Asn Arg Leu Ala Asp Leu
Ile 100 105 110 Glu Arg Asp Arg Thr Tyr Leu Ala Ala Leu Glu Thr Leu
Asp Asn Gly 115 120 125 Lys Pro Tyr Val Ile Ser Tyr Leu Val Asp Leu
Asp Met Val Leu Lys 130 135 140 Cys Leu Arg Tyr Tyr Ala Gly Trp Ala
Asp Lys Tyr His Gly Lys Thr 145 150 155 160 Ile Pro Ile Asp Gly Asp
Phe Phe Ser Tyr Thr Arg His Glu Pro Val 165 170 175 Gly Val Cys Gly
Gln Ile Ile Pro Trp Asn Phe Pro Leu Leu Met Gln 180 185 190 Ala Trp
Lys Leu Gly Pro Ala Leu Ala Thr Gly Asn Val Val Val Met 195 200 205
Lys Val Ala Glu Gln Thr Pro Leu Thr Ala Leu Tyr Val Ala Asn Leu 210
215 220 Ile Lys Glu Ala Gly Phe Pro Pro Gly Val Val Asn Ile Val Pro
Gly 225 230 235 240 Phe Gly Pro Thr Ala Gly Ala Ala Ile Ala Ser His
Glu Asp Val Asp 245 250 255 Lys Val Ala Phe Thr Gly Ser Thr Glu Ile
Gly Arg Val Ile Gln Val 260 265 270 Ala Ala Gly Ser Ser Asn Leu Lys
Arg Val Thr Leu Glu Leu Gly Gly 275 280 285 Lys Ser Pro Asn Ile Ile
Met Ser
Asp Ala Asp Met Asp Trp Ala Val 290 295 300 Glu Gln Ala His Phe Ala
Leu Phe Phe Asn Gln Gly Gln Cys Cys Cys 305 310 315 320 Ala Gly Ser
Arg Thr Phe Val Gln Glu Asp Ile Tyr Asp Glu Phe Val 325 330 335 Glu
Arg Ser Val Ala Arg Ala Lys Ser Arg Val Val Gly Asn Pro Phe 340 345
350 Asp Ser Lys Thr Glu Gln Gly Pro Gln Val Asp Glu Thr Gln Phe Lys
355 360 365 Lys Ile Leu Gly Tyr Ile Asn Thr Gly Lys Gln Glu Gly Ala
Lys Leu 370 375 380 Leu Cys Gly Gly Gly Ile Ala Ala Asp Arg Gly Tyr
Phe Ile Gln Pro 385 390 395 400 Thr Val Phe Gly Asp Val Gln Asp Gly
Met Thr Ile Ala Lys Glu Glu 405 410 415 Ile Phe Gly Pro Val Met Gln
Ile Leu Lys Phe Lys Thr Ile Glu Glu 420 425 430 Val Val Gly Arg Ala
Asn Asn Ser Thr Tyr Gly Leu Ala Ala Ala Val 435 440 445 Phe Thr Lys
Asp Leu Asp Lys Ala Asn Tyr Leu Ser Gln Ala Leu Gln 450 455 460 Ala
Gly Thr Val Trp Val Asn Cys Tyr Asp Val Phe Gly Ala Gln Ser 465 470
475 480 Pro Phe Gly Gly Tyr Lys Met Ser Gly Ser Gly Arg Glu Leu Gly
Glu 485 490 495 Tyr Gly Leu Gln Ala Tyr Thr Glu Val Lys Thr Val Thr
Val Lys Val 500 505 510 Pro Gln Lys Asn Ser 515 55443PRTHomo
sapiens 55Met Ala Ala Ser Pro His Thr Leu Ser Ser Arg Leu Leu Thr
Gly Cys 1 5 10 15 Val Gly Gly Ser Val Trp Tyr Leu Glu Arg Arg Thr
Ile Gln Asp Ser 20 25 30 Pro His Lys Phe Leu His Leu Leu Arg Asn
Val Asn Lys Gln Trp Ile 35 40 45 Thr Phe Gln His Phe Ser Phe Leu
Lys Arg Met Tyr Val Thr Gln Leu 50 55 60 Asn Arg Ser His Asn Gln
Gln Val Arg Pro Lys Pro Glu Pro Val Ala 65 70 75 80 Ser Pro Phe Leu
Glu Lys Thr Ser Ser Gly Gln Ala Lys Ala Glu Ile 85 90 95 Tyr Glu
Met Arg Pro Leu Ser Pro Pro Ser Leu Ser Leu Ser Arg Lys 100 105 110
Pro Asn Glu Lys Glu Leu Ile Glu Leu Glu Pro Asp Ser Val Ile Glu 115
120 125 Asp Ser Ile Asp Val Gly Lys Glu Thr Lys Glu Glu Lys Arg Trp
Lys 130 135 140 Glu Met Lys Leu Gln Val Tyr Asp Leu Pro Gly Ile Leu
Ala Arg Leu 145 150 155 160 Ser Lys Ile Lys Leu Thr Ala Leu Val Val
Ser Thr Thr Ala Ala Gly 165 170 175 Phe Ala Leu Ala Pro Gly Pro Phe
Asp Trp Pro Cys Phe Leu Leu Thr 180 185 190 Ser Val Gly Thr Gly Leu
Ala Ser Cys Ala Ala Asn Ser Ile Asn Gln 195 200 205 Phe Phe Glu Val
Pro Phe Asp Ser Asn Met Asn Arg Thr Lys Asn Arg 210 215 220 Pro Leu
Val Arg Gly Gln Ile Ser Pro Leu Leu Ala Val Ser Phe Ala 225 230 235
240 Thr Cys Cys Ala Val Pro Gly Val Ala Ile Leu Thr Leu Gly Val Asn
245 250 255 Pro Leu Thr Gly Ala Leu Gly Leu Phe Asn Ile Phe Leu Tyr
Thr Cys 260 265 270 Cys Tyr Thr Pro Leu Lys Arg Ile Ser Ile Ala Asn
Thr Trp Val Gly 275 280 285 Ala Val Val Gly Ala Ile Pro Pro Val Met
Gly Trp Thr Ala Ala Thr 290 295 300 Gly Ser Leu Asp Ala Gly Ala Phe
Leu Leu Gly Gly Ile Leu Tyr Ser 305 310 315 320 Trp Gln Phe Pro His
Phe Asn Ala Leu Ser Trp Gly Leu Arg Glu Asp 325 330 335 Tyr Ser Arg
Gly Gly Tyr Cys Met Met Ser Val Thr His Pro Gly Leu 340 345 350 Cys
Arg Arg Val Ala Leu Arg His Cys Leu Ala Leu Leu Val Leu Ser 355 360
365 Ala Ala Ala Pro Val Leu Asp Ile Thr Thr Trp Thr Phe Pro Ile Met
370 375 380 Ala Leu Pro Ile Asn Ala Tyr Ile Ser Tyr Leu Gly Phe Arg
Phe Tyr 385 390 395 400 Val Asp Ala Asp Arg Arg Ser Ser Arg Arg Leu
Phe Phe Cys Ser Leu 405 410 415 Trp His Leu Pro Leu Leu Leu Leu Leu
Met Leu Thr Cys Lys Arg Pro 420 425 430 Ser Gly Gly Gly Asp Ala Gly
Pro Pro Pro Ser 435 440 56239PRTHomo sapiens 56Met Ala Pro Ser Val
Pro Ala Ala Glu Pro Glu Tyr Pro Lys Gly Ile 1 5 10 15 Arg Ala Val
Leu Leu Gly Pro Pro Gly Ala Gly Lys Gly Thr Gln Ala 20 25 30 Pro
Arg Leu Ala Glu Asn Phe Cys Val Cys His Leu Ala Thr Gly Asp 35 40
45 Met Leu Arg Ala Met Val Ala Ser Gly Ser Glu Leu Gly Lys Lys Leu
50 55 60 Lys Ala Thr Met Asp Ala Gly Lys Leu Val Ser Asp Glu Met
Val Val 65 70 75 80 Glu Leu Ile Glu Lys Asn Leu Glu Thr Pro Leu Cys
Lys Asn Gly Phe 85 90 95 Leu Leu Asp Gly Phe Pro Arg Thr Val Arg
Gln Ala Glu Met Leu Asp 100 105 110 Asp Leu Met Glu Lys Arg Lys Glu
Lys Leu Asp Ser Val Ile Glu Phe 115 120 125 Ser Ile Pro Asp Ser Leu
Leu Ile Arg Arg Ile Thr Gly Arg Leu Ile 130 135 140 His Pro Lys Ser
Gly Arg Ser Tyr His Glu Glu Phe Asn Pro Pro Lys 145 150 155 160 Glu
Pro Met Lys Asp Asp Ile Thr Gly Glu Pro Leu Ile Arg Arg Ser 165 170
175 Asp Asp Asn Glu Lys Ala Leu Lys Ile Arg Leu Gln Ala Tyr His Thr
180 185 190 Gln Thr Thr Pro Leu Ile Glu Tyr Tyr Arg Lys Arg Gly Ile
His Ser 195 200 205 Ala Ile Asp Ala Ser Gln Thr Pro Asp Val Val Phe
Ala Ser Ile Leu 210 215 220 Ala Ala Phe Ser Lys Ala Thr Cys Lys Asp
Leu Val Met Phe Ile 225 230 235 57922DNAHomo sapiens 57tatcagaagg
ccaggcgaga ctgcaacact gctcatcacc ccgcggcgtg atccctgctc 60ttaggtgctg
ggcagagggg aagggtggtc agggtgagga tggtgaggga gggctggtga
120ggggctcaga ggaatacttg gaacaatagc agtgttattg tagtgtggca
gtttctttta 180tacataggtg agagttttta aagtgtaagg gaaaaattaa
ttttttaaaa aacaccatgc 240ttggagggtg ggggtagaaa tagacacaat
attatttcta aggaatcggg ttttcattta 300ctctggactg gtgaaaatat
tttttaaagc cagtgctcta agacctcagc ttttatctca 360gaaccccatg
ggttccagac caagagtaca ggaaatcaaa ttgttgtcct gtctgtctat
420agcttggaac agggagcttt gattactgac tccggttcca cacactgtaa
gatcaaaaac 480catctccaca tttgaaagag atgtaaggtg tattcatagg
gatggtggct caacaaatca 540agcaaactgg aatcaagggg agggggaagg
gaatgaaatg gaaagggagg ctgattccct 600tcccctgact taccactaat
ttactaggct acctactttc atgagtaacc tctcacagct 660acccagcaca
tgccacaatc ctatgctctt gccttctttt atctgcactg tgtgaaggga
720ctcttttaaa taaatgagca agtgtcctaa gctatgtcat ccaaagattg
tcctttccat 780tctcaaatcc tgtgactggg atcactcaac agcactgtga
tgtattattt tcaatgaggt 840gccttctaaa actgaccaaa tgctgccttg
tttggcccct aaatcaataa aatatgttaa 900aatttgaaaa aaaaaaaaaa aa
9225833DNAArtificial SequenceDescription of Artificial Sequence
synthetic Primer 58cgactacggc gccctggaac ctcacatcaa cgc
33591425DNAHomo sapiens 59gtgttgatgt atcacctccc caaaactgtt
ggtaaatgtc agattttttc ctccaagagt 60tgtgcttttg tgttatttgt tttcactcaa
atattttgcc tcattattct tgttttaaaa 120gaaagaaaac aggccgggca
cagtggctca tgcctgtaat cccagcactt tgggaggtcg 180aggtgggtgg
atcacttggg gtcagggttt gagaccagcc tggccaacat ggcggaaccc
240tgtctctacc aaaattacaa aaattagccg agcatggtgg cgcatgcctg
tagtcgcagc 300tactcgcgag gttgaggcag gagaattgct tgaacccagg
aagtggcagt tgcagtgagc 360cgagacgaca ccactgcact ccagcctggg
tgacagaggg agactctgtc tcgaaagaaa 420gaaagaaaaa aaggaaggaa
ggagaaggaa ggaaggagaa gaaaaggtac ctgttctacg 480tagaacacct
ttggtggagt tccatcaact cgcaaagtag aatccttacc tactactctt
540ctgataataa ttttaatatt ttttatgttt ggttgatgcg agcagctgca
ctgctcatgc 600agttagctag catgtgacat catgtgacaa agttcatgta
attagatgga agaaacctca 660ctgattaatt ttaagaacct tttagggatg
caggaacaat gaagtggcca cagtatgtgc 720tgtttttgaa gcatttttaa
aaacgaattg tagttgtttt tcttcattta aaatggatct 780gttggaggtt
atgtgtgtat gttgtagttt tattgcagcc acaataattt taccaaagtt
840ttcacatagg cagttagcct ttacttaata tcaagacaag tgaaaaaata
ttggcatcga 900tgaaaccgat aacattggcc tcattggatt tctttaccca
ttcacagtgt aaagaagtta 960ccttcatgct ttcattgtac ctgcaggcct
gtgggcttgt acagtagata attaatttct 1020aaaaagaaca gctgcctatt
ttcttcctag gttaggttat atcttcataa tcacaagaat 1080tagtgatggc
aaaataaaat tttgcttatg aatcttttac attgtttata tatgattaat
1140atcatcatat atattttctg tattaagctc atttggcttc atttaagctg
tatacttagt 1200catatatctt tcattagttc tatggatatg agcagatccc
tttactggag cccagtatgt 1260gctgtgtgag ttagaagtca ttcttgctga
gaaggtgaat aggtagggat ttgccttgtt 1320ttgtaagtct acaatttgcc
aagagtaaat aacactggac cagctgtaaa agtaaacagt 1380gtgtttatgc
attgagatac taaagcattt aagaaaaaat taaaa 142560202DNAHomo sapiens
60ttatgctgag tatgttaagc tctttatgac tgtttttgta gtggtataga gtactgcaga
60atacagtaag ctgctctatt gtagcatttc ctgatgttgc ttagtcactt atttcataaa
120caacttaatg ttctgaataa tttcttacta aacattttgt tattgggcaa
gtgattgaaa 180atagtaaatg ctttgtgtga tt 202
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