U.S. patent application number 17/447372 was filed with the patent office on 2022-01-06 for factor ix polypeptide mutant, its uses and a method for its production.
This patent application is currently assigned to uniQure biopharma B.V.. The applicant listed for this patent is uniQure biopharma B.V.. Invention is credited to Paolo SIMIONI.
Application Number | 20220002702 17/447372 |
Document ID | / |
Family ID | 1000005827971 |
Filed Date | 2022-01-06 |
United States Patent
Application |
20220002702 |
Kind Code |
A1 |
SIMIONI; Paolo |
January 6, 2022 |
FACTOR IX POLYPEPTIDE MUTANT, ITS USES AND A METHOD FOR ITS
PRODUCTION
Abstract
Disclosed are a modified FIX (Factor IX) polypeptide comprising
a leucine, cysteine, aspartic acid, glutamic acid, histidine,
lysine, asparagine, glutamine or tyrosine in position 338;
pharmaceutical preparations containing said modified FIX
polypeptide; a nucleotide sequence coding for the modified FIX
polypeptide; and a method for producing the modified FIX
polypeptide.
Inventors: |
SIMIONI; Paolo; (Padova,
IT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
uniQure biopharma B.V. |
Amsterdam |
|
NL |
|
|
Assignee: |
uniQure biopharma B.V.
Amsterdam
NL
|
Family ID: |
1000005827971 |
Appl. No.: |
17/447372 |
Filed: |
September 10, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16707414 |
Dec 9, 2019 |
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17447372 |
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16589851 |
Oct 1, 2019 |
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16707414 |
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15989665 |
May 25, 2018 |
10465180 |
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16589851 |
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15650070 |
Jul 14, 2017 |
9982248 |
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15989665 |
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14981981 |
Dec 29, 2015 |
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15650070 |
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13063898 |
May 31, 2011 |
9249405 |
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PCT/EP2009/061935 |
Sep 15, 2009 |
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14981981 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Y 304/21022 20130101;
A61K 38/4846 20130101; C12N 9/644 20130101 |
International
Class: |
C12N 9/64 20060101
C12N009/64; A61K 38/48 20060101 A61K038/48 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 15, 2008 |
IT |
BO2008A000564 |
May 6, 2009 |
IT |
BO2009A000275 |
Claims
1. An adeno-associated virus vector comprising: (a) a nucleic acid
encoding a modified FIX polypeptide, the modified FIX polypeptide
comprising SEQ ID NO:1 having a leucine at position 384 of SEQ ID
NO:1 and an alanine or threonine at position 194 of SEQ ID NO:1;
and (b) a promoter sequence.
2. A pharmaceutical composition comprising the adeno-associated
virus vector of claim 1.
3. A method of treatment of at least one coagulopathy in a patient
in need thereof comprising administering the adeno-associated virus
vector of claim 1 to the patient.
4. The method of claim 3, wherein the at least one coagulopathy is
haemophilia B.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/707,414 filed Dec. 9, 2019, which is a
continuation of U.S. patent application Ser. No. 16/589,851 filed
Oct. 1, 2019, which is a continuation of U.S. patent application
Ser. No. 15/989,665 filed May 25, 2018 now U.S. Pat. No. 10,465,180
issued Nov. 5, 2019, which is a continuation of U.S. patent
application Ser. No. 15/650,070 filed Jul. 14, 2017, now U.S. Pat.
No. 9,982,248 issued May 29, 2018, which is a continuation of U.S.
patent application Ser. No. 14/981,981 filed Dec. 29, 2015, now
abandoned, which is a continuation of U.S. patent application Ser.
No. 13/063,898 filed May 31, 2011, now U.S. Pat. No. 9,249,405
issued Feb. 2, 2016, which is a 371 national stage of International
Application No. PCT/EP2009/061935 filed Sep. 15, 2009, which claim
the benefit of Italian Application Nos. BO2009A000275 filed May 6,
2009 and BO2008A000564 filed Sep. 15, 2008. All of the foregoing
are incorporated by reference in their entireties.
FIELD OF THE INVENTION
[0002] The present invention relates to a modified AX (factor IX)
polypeptide, a nucleotide sequence, a vector comprising said
nucleotide sequence and a method for producing the modified AX
polypeptide.
[0003] The present invention also relates to pharmaceutical
preparations and uses of modified factor FIX and of the nucleotide
sequence.
PRIOR ART
[0004] FIX is a vitamin K-dependent glycoprotein belonging to the
serine-protease family, and is synthesized in the liver of man and
other animals, including mammals, playing a fundamental role in
both intrinsic and extrinsic pathways of the coagulation cascade.
Human FIX circulates in plasma as a single chain zymogen composed
of 415 amino acids. Human FIX has a molecular weight of 56 kD and a
plasma concentration of about 5 pg/ml. The zymogen is activated
both by activated factor XI (FXIa), and tissue factor complex
(TF)--activated factor VII (FVIIa). The structural organization of
FIX is similar to that of other vitamin K-dependent coagulation
proteins such as factor VII (FV1I), factor X (FX) and protein C
(PC). The amino-terminal portion of the molecule comprises the
"Gla" domain, a region rich in gamma-carboxy-glutamic residues
whose carboxylation is dependent on the presence of vitamin K. The
main physiological function of FIX, once activated, is to convert
factor X (FX) into activated factor X (FXa) in a process that
requires the presence of a phospholipid surface, calcium ions and a
protein with cofactor effect, namely activated factor VIII
(FVIIIa). FXa itself is able to convert prothrombin into thrombin
which transforms fibrinogen into soluble fibrin which, on
polymerization, forms the clot. The action of FXa is enhanced by
the presence of activated factor V (FVa).
[0005] The human FIX gene is located on chromosome X in position
Xq27.1 and contains 8 exons of lengths varying from 25 base pairs
(bp) to 2000 bp. Human FIX mRNA is about 3 kb in length and
comprises 205 bases which form the 5' UTR region, 1386 bases which
encode the FIX polypeptide and 1392 bases of the 3' UTR region.
This mRNA encodes the synthesis of 461 amino acids which form the
human FIX precursor. This precursor (SEQ ID NO: 1) comprises the
following segments and domains: a hydrophobic signal peptide (amino
acids 1-28), a propeptide (amino acids 29-46), a Gla-domain (amino
acids 47 to 92), an EGF-like 1 domain (amino acids 93 to 129), an
EGF-like 2 domain (amino acids 130 to 171), an activation peptide
(amino acids 192 to 226) and a serine-protease domain (amino acids
227 to 461). The mature form of human FIX (SEQ ID NO: 2) loses the
hydrophobic signal peptide and the propeptide. Consequently the
corresponding amino acid positions of the aforementioned domains
become the following: a Gla-domain (amino acids 1 to 46), an
EGF-like 1 domain (amino acids 47 to 83), an EGF-like 2 domain
(amino acids 84 to 125), an activation peptide (amino acids 146 to
180) and a serine-protease domain (amino acids 181 to 415). SEQ ID
NO: 1 (from which SEQ ID NO: 2 is derived) corresponds to the
sequence on PubMed ("Protein" category) found by entering accession
number AAB59620; this amino acid sequence comprises the signal
peptide (46 AA), followed by the amino acid sequence of the mature
protein.
[0006] A genetic deficiency in FIX can cause a number of
coagulation diseases (coagulopathies), for example the haemorrhagic
disease known as haemophilia B in affected males (sex linked
genetic disease). Haemophilia B can be classified into three
classes, each of which is characterized by the presence of
different plasma concentrations of FIX. In severe haemophilia B the
plasma levels of FIX activity are below 1% of normal; in the
moderate form, levels are between 1% and 5%; in the mild form,
between 5 and 25% of normal levels. There are also healthy carrier
individuals who have medium FIX activity levels, between 25% and
50% of normal, but many carriers can have levels even exceeding
50%. Patients affected by severe haemophilia B present serious
haemorrhagic manifestations which can be controlled or avoided by
administering FIX concentrates of extractive (from human plasma) or
of recombinant origin, currently only available in a single
commercial formulation.
[0007] Attempts to correct the genetic defect by means of gene
therapy have so far been fruitless because of various problems.
These include firstly those connected to the low efficiency of
expression in man of FIX levels in plasma i.e. around 1%, hence not
sufficient to correct the disease; those connected to the
immunogenicity of treatment with viral vectors; finally those
connected to the side effects of gene therapy itself which include
hepatitis, myositis and others.
[0008] An increase in plasma FIX to higher than normal levels
(normal range of FIX in plasma being 70-120% i.e. 70-120 U/dl,
where a unit is the quantity of FIX contained in 1 millilitre of
normal plasma, equal to about 5 .mu.g) has been associated with an
increased risk in humans of developing thrombotic manifestations in
the venous system. In particular, for values above 150 U/dl, a 4.8
fold increase in thrombotic risk has been noted (corrected 0. R.
4.8; 95% CI, from 2.3 to 10.1). However, the genetic basis for the
increased FIX levels in plasma of these individuals has never been
identified.
[0009] In vitro mutagenesis studies of mutated recombinant FIX
expression have demonstrated the possibility of reproducing the
alterations in FIX synthesis and activity encountered in vivo in
patients with haemophilia B. Vice-versa, by site-specific
mutagenesis in certain positions on the FIX molecule, FIX mutants
have been produced with "gain-of-function" (increased activity
relative to the normal molecule) by altering their specificity for
physiological substrates and/or modifying their other functions. In
WO 99/03496 is disclosed the recombinant FIX arginine 338 alanine
mutant which resulted in a gain-of-function whose activity levels
are 2-3 folds higher than that found in wild type FIX. These
gain-of-function mutants (in particular with increased protease
activity towards the physiological substrate, i.e. FX, or with an
increased capacity for interaction with FVllla, a cofactor of FIXa)
have not as yet been found to exist in nature, nor have they been
tested in man. More explicitly, there is no evidence of: 1) the
existence of a human carrier of mutated FIX (natural FIX mutant)
with gain-of-function characterized by increased functional
activity as compared to normal FIX (WT) with any gain-of-function
in functional activity; 2) tests conducted in vivo in man with
administrations of modified recombinant FIX; 3) tests conducted in
vivo in man with administrations of modified recombinant FIX with
gain-of-function for the prophylaxis and treatment of patients
affected by haemophilia (genetic or acquired) or other
coagulopathies; 4) tests conducted in vivo in man with
administrations of modified recombinant FIX which show the absence
of side effects.
SUMMARY OF THE INVENTION
[0010] The object of the present invention is to provide a modified
FIX polypeptide, a nucleotide sequence, a vector comprising said
nucleotide sequence, and a method for producing the modified FIX
polypeptide.
[0011] A further object of the present invention is to provide
pharmaceutical preparations and uses for modified factor FIX and
the nucleotide sequence.
DETAILED DESCRIPTION OF THE INVENTION
[0012] According to the present invention are provided
polypeptides, nucleotide sequences, vectors, a method of
production, uses of the polypeptides and nucleotide sequences and
the pharmaceutical preparations, according to that described in the
following independent claims and preferably in any one of the
claims that depend directly or indirectly on the independent
claims.
[0013] The modified FIX polypeptides herein described show a
gain-of-function of at least 5 folds higher than that of the
wild-type FIX molecule. This increase in the activity level is
unexpectedly even higher than that disclosed for the known
recombinant FIX arginine 338 alanine mutant.
[0014] The contents of the references (articles, textbooks, GenBank
sequences etc.) cited in the present text are fully included herein
for descriptive completion. In particular, the references
(articles, textbooks, GenBank sequences etc.) cited in the present
text are incorporated herein for reference.
[0015] Unless otherwise explicitly specified, the following terms
have the meanings indicated below.
[0016] In the present text the term "percentage identity" and "%
identity" between two amino acid (peptide) or nucleic acid
(nucleotide) sequences means the percentage of identical amino acid
or nucleotide residues in corresponding positions in the two
optimally aligned sequences.
[0017] To determine the "percentage identity" of the two amino acid
or nucleic acid sequences, the sequences are aligned together. To
achieve an optimal match, gaps can be introduced into the sequence
(i.e. deletions or insertions which can also be placed at the
sequence ends). Amino acid and nucleotide residues in the
corresponding positions are then compared. When a position in the
first sequence is occupied by the same amino acid or nucleotide
residue that occupies the corresponding position in the second
sequence, the molecules are identical in that position. The
percentage identity between two sequences is a function of the
number of identical positions divided by the sequences [i.e. %
identity=(number of identical positions/total number of
positions).times.100]
[0018] According to an advantageous embodiment, the sequences have
the same length. Advantageously, the compared sequences do not have
gaps (or insertions).
[0019] The percentage identity can be obtained by using
mathematical algorithms. A non-limiting example of an algorithm
used for comparing two sequences is the Karlin and Altschul
algorithm [Proc. Natl. Acad. Sci. USA 87 (1990) 2264-2268] modified
by Karlin and Altschul [Proc. Natl. Acad. Sci. USA 90 (1993)
5873-5877]. Said algorithm is incorporated in the BLASTn and BLASTp
programmes of Altschul [Altschul et al, J. Mol. Bio. 215 (1990)
403-410].
[0020] With the purpose of achieving alignments even in the
presence of one or more gaps (or insertions) methods may be used
which assign a relatively high penalty for each gap (or insertion)
and a lower penalty for each additional amino acid or nucleotide
residue in the gap (this additional amino acid or nucleotide
residue is defined as gap extension). High penalties will obviously
lead to the alignments being optimized with the least number of
gaps.
[0021] An example of a programme able to achieve this type of
alignment is the BLAST programme as described in Altschul et al.,
Nucleic Acids Res. 25 (1997) 3389-3402. For this purpose the BLASTn
and BLASTp programmes can be used with the default parameters. When
using the BLAST programme the BLOSUM62 matrix is typically
employed.
[0022] An advantageous and non-limiting example of a programme for
achieving an optimal alignment is GCG Wisconsin Bestfit package
(University of Wisconsin, USA; Devereux et. al., 1984, Nucleic Acid
Research 12:387). The default parameters are again used i.e. for an
amino acid sequence they allow a penalty of -12 for a gap and a
penalty of -4 for each extension.
[0023] In the present text the term "percentage homology" and "%
homology" between two amino acid or nucleotide sequences means the
percentage of homologous amino acid or nucleotide residues in
corresponding positions in the two optimally aligned sequences.
[0024] The percentage homology between two sequences is determined
in a substantially identical manner to that described above for
determining percentage identity except for the fact that homologous
positions and not only identical positions are considered in the
calculation.
[0025] With regard to a nucleotide sequence, two homologous
positions present two different nucleotides but which, within their
codon, code for the same amino acid. With regard to an amino acid
sequence, two homologous positions present two homologous amino
acids, that is to say amino acids possessing similar
physicochemical properties, for example amino acids belonging to
the same groups such as: aromatic (Phe, Trp, Tyr), acids (Glu,
Asp), polar (Gln, Asn), basic (Lys, Arg, His), aliphatic (Ala, Leu,
Ile, Val), with a hydroxyl group (Ser, Thr), with a short side
chain (Gly, Ala, Ser, Thr, Met). It is expected that substitutions
between these homologous amino acids would not change the phenotype
of the proteins (conservative amino acid substitutions). Specific
examples of conservative substitutions are known in this technical
field and are described in the various literature (e.g. Bowie et
al., Science, 247:1306-1310 (1990)).
[0026] Further examples of programmes and/or articles relating to
the determination of alignments and percentage homologies and/or
identities are indicated in, for example, US2008003202,
US2007093443, W006048777.
[0027] In the present text the term "corresponding position" means
a position in a polypeptide or nucleic acid sequence which,
following an alignment, corresponds to (or faces), a precise
position in a reference sequence. For example, a position
corresponding to a precise position on the FIX polypeptide
presenting SEQ ID NO: 2 can be determined by aligning the SEQ ID
NO: 2 with a polypeptide of interest; the alignment can be carried
out manually or as explained above in relation to percentage
identity determination.
[0028] In the present text the term "naked chain" means a
polypeptide which has not been chemically modified but contains
only covalently bound amino acids.
[0029] In the present text the term "promoter" means a DNA portion
of a gene that controls (activates) the transcription of a
nucleotide sequence to which it is operatively linked (but not
necessarily flanking it). The promoter includes one or more DNA
sequences, which are recognized by RNA polymerase and bind RNA
polymerase so that RNA polymerase itself initiates
transcription.
[0030] In the present text the term "treat" or "treatment" of a
pathology means the prophylaxis and/or therapy and/or cure of this
pathology. The term prophylaxis means advantageously to at least
partially arrest the development of a potential disease and/or to
prevent the worsening of symptoms or progression of a disease.
Advantageously, the term therapy means a partial or total
alleviation of the disease symptoms.
[0031] In the present text the term "vector" means an element used
to introduce a nucleic acid into a cell for the expression or
replication of said nucleic acid. An example of vectors are
episomes, which are capable of extra-chromosomal replication. The
vectors can also be integrated into host chromosomes. Vectors are
often in the form of plasmids, generally circular double-helical
DNA.
[0032] In the present text "vehicle presenting a nucleic acid"
means: a vector which includes nucleic acid; a cell which includes
nucleic acid; or a pharmaceutically acceptable excipient combined
with the nucleic acid by mixing. Advantageously the vehicle is
chosen from a vector or a cell.
[0033] The invention will now be described with reference to the
accompanying drawing which illustrates a non-limiting example of
its implementation, in which:
[0034] FIG. 1 illustrates an SDS-PAGE and immunoblot of a normal
FIX polypeptide (2), a modified FIX polypeptide according to the
present invention (3), a recombinant modified FIX polypeptide
according to the present invention (4).
[0035] According to a first aspect of the present invention, a
modified FIX polypeptide is provided comprising an amino acid
chosen from the group consisting of: leucine, cysteine, aspartic
acid. glutamic acid. histidine, lysine, asparagine, glutamine,
tyrosine in a position corresponding to position 338.
[0036] According to other embodiments, the amino acid is chosen
from the group consisting of: leucine, aspartic acid,
glutamine.
[0037] According to other embodiments, the amino acid is chosen
from the group consisting of: aspartic acid, glutamine.
[0038] According to other embodiments, the amino acid is aspartic
acid.
[0039] According to other embodiments, the amino acid is
glutamine.
[0040] According to other embodiments, the amino acid is chosen
from the group consisting of: aspartic acid, leucine.
[0041] According to other embodiments, the amino acid is chosen
from the group consisting of: leucine, glutamine.
[0042] According to other embodiments, the amino acid is
leucine.
[0043] The modified FIX polypeptide must be able to carry out its
function within the coagulation cascade and can be of synthetic or
natural origin, for example human or animal origin.
[0044] Examples of FIX polypeptides include (but are not limited
to) unmodified wild-type FIX (such as the polypeptide of SEQ ID NO:
2), precursors of said wild-type FIX (such as the polypeptide of
SEQ ID NO: 1), natural polymorphic variants (such as: a polypeptide
presenting an alanine in a position corresponding to position T148
or to a precursor polypeptide thereof).
[0045] In the present text the loci (positions) of the modified or
unmodified amino acid sequences are identified by reference to the
amino acid numbering in the corresponding positions of an
unmodified mature FIX polypeptide, as identified by SEQ ID NO: 2.
Corresponding positions can be determined by alignment of
unmodified residues (see above). By way of example we report
hereinafter the sequences and relative numberings of the mature FIX
polypeptide (SEQ ID NO: 2) and of the FIX polypeptide precursor
(SEQ ID NO:1).
TABLE-US-00001 SEQ ID NO: 1 MQRVNMIMAE SPGLITICLL GYLLSAECTV
FLDHENANKI LNRPKRYNSG KLEEFVQGNL ERECMEEKCS FEEAREVFEN TERTTEFWKQ
YVDGDQCESN PCLNGGSCKD DINSYECWCP FGFEGKNCEL DVTCNIKNGR CEQFCKNSAD
NKVVCSCTEG YRLAENQKSC EPAVPFPCGR VSVSQTSKLT RAEAVFPDVD YVNSTEAETI
LDNITQSTQS FNDFTRVVGG EDAKPGQFPW QVVLNGKVDA FCGGSIVNEK WIVTAAHCVE
TGVKITVVAG EHNIEETEHT EQKRNVIRII PHHNYNAAIN KYNHDIALLE LDEPLVLNSY
VTPICIADKE YTNIFLKFGS GYVSGWGRVF HKGRSALVLQ YLRVPLVDRA TCLRSTKFTI
YNNMFCAGFH EGGRDSCQGD SGGPHVTEVE GTSFLTGIIS WGEECAMKGK YGIYTKVSRY
VNWIKEKTKL T
in bold underlined Arg 384 corresponding to Arg 338 in SEQ ID NO:
2
TABLE-US-00002 SEQ ID NO: 2 YNSGKLEEFV QGNLERECME EKCSFEEARE
VFENTERTTE FWKQYVDGDQ CESNPCLNGG SCKDDINSYE CWCPFGFEGK NCELDVTCNI
KNGRCEQFCK NSADNKVVCS CTEGYRLAEN QKSCEPAVPF PCGRVSVSQT SKLTRAETVF
PDVDYVNSTE AETILDNITQ STQSFNDFTR VVGGEDAKPG QFPWQVVLNG KVDAFCGGSI
VNEKWIVTAA HCVETGVKIT VVAGEHNIEE TEHTEQKRNV IRIIPHHNYN AAINKYNHDI
ALLELDEPLV LNSYVTPICI ADKEYTNIFL KFGSGYVSGW GRVFHKGRSA LVLQYLRVPL
VDRATCLRST KFTIYNNMFC AGFHEGGRDS CQGDSGGPHV TEVEGTSFLT GIISWGEECA
MKGKYGIYTK VSRYVNWIKE KTKLT
in bold underlined Arg 338.
[0046] Likewise, the positions of the modified or unmodified
nucleotide sequences are identified, unless otherwise indicated, by
reference to the nucleotide numbering in the corresponding
positions of the nucleotide sequence identified by accession number
K02402(GenBank). The nucleotide sequence K02402 codes for the AX
polypeptide precursor (SEQ ID NO: 1) and includes some intron
regions (in this regard see Anson D S, Choo K H, Rees D J,
Giannelli F, Gould K, Huddleston J A, Brownlee G G. The gene
structure of human anti-haemophilic factor IX. The EMBO Journal
1984;3:1053-1060).
[0047] Included within the definition of a modified FIX polypeptide
are chimeric variants which can be produced by replacing amino
acids or entire domains of the FIX with amino acids or sequences of
other factors belonging to the coagulation factor family (for
example factor VII or factor X).
[0048] According to other embodiments, the modified FIX polypeptide
presented herein is either a naked chain or exhibits
post-transcriptional modifications. Examples of modifications
include one or more chemical modifications, which comprise (but are
not limited to): glycosylation, acylation, methylation,
phosphorylation, sulphation, carboxylation, salification, vitamin
C-dependent modifications such as hydrolysis of proline, aspartic
acid, lysine, or carboxy-terminal amidation; vitamin K-dependent
modifications such as carboxylation of glutamic acid residues;
incorporation of selenium to form one or more selenocysteine(s);
incorporation of a PEG moiety (polyethylene glycol).
[0049] In addition to the possible modifications disclosed herein,
the modified FIX polypeptide can contain one or more variants known
in the state of the art such as hyperglycosylation, deimmunization
and others (see for example: U.S. Pat. Nos. 6,277,618, 6,315,995,
6,531,298, US2004/0102388, US2004/0110675, US2004/0254106,
U52005/0100982, US2006/0040856).
[0050] Non-limiting examples of modified FIX polypeptide variants
can be deduced from one or more of the following references:
US2006/040856, Friedler et al (2000) J. Biol Chem. 275:23783-23789,
US2004/102388, W02006/018201, Lim et al. (1990) J. Biol Chem.
265(1):144-150, Cheung et al. (1992) J. Biol. Chem. 267(29):
20529-20531, Gui et al. (2002) Blood 100(1)1 53-158, Schuettrumpf
et al. (2005) Blood 105(6): 2316-2323, US2004/110675, U.S. Pat. No.
6,315,995.
[0051] According to some alternative embodiments, the modified FIX
polypeptide has at least 50%, 60%, 70%, 80%, 85%, 90%, 94%, 97%,
99%, 100% homology (or, advantageously, identity) with a peptide
sequence chosen from the group consisting of: SEQ ID NO: 1 and SEQ
ID NO: 2.
[0052] Advantageously, the modified FIX polypeptide has at least
60% homology (or, advantageously, identity) with a peptide sequence
chosen from the group consisting of: SEQ ID NO: 1 and SEQ ID NO:
2.
[0053] Advantageously, the modified FIX polypeptide has at least
80% homology (or, advantageously, identity) with a peptide sequence
chosen from the group consisting of: SEQ ID NO: 1 and SEQ ID NO:
2.
[0054] Advantageously, the modified FIX polypeptide has at least
90% homology (or, advantageously, identity) with a peptide sequence
chosen from the group consisting of: SEQ ID NO: 1 and SEQ ID NO:
2.
[0055] Advantageously, the peptide sequence is SEQ ID NO: 2.
[0056] According to a second aspect of the present invention, a
nucleotide sequence is provided which codes for the FIX polypeptide
of the first aspect of the present invention.
[0057] According to some alternative embodiments, the nucleotide
sequence has at least 50%, 60%, 70%, 80%, 85%, 90%, 94%, 97%, 99%,
100% homology (or, advantageously, identity) with the sequence
having accession number K02402 (GenBank).
[0058] Advantageously, the nucleotide sequence has at least 70%
homology (or, advantageously, identity) with the sequence having
accession number K02402 (GenBank).
[0059] Advantageously, the nucleotide sequence has at least 90%
homology (or, identity) with the sequence having accession number
K02402 (GenBank).
[0060] Advantageously, the nucleotide sequence has at least 100%
homology (or, advantageously, identity) with the sequence having
accession number K02402 (GenBank).
[0061] According to some alternative embodiments, the nucleotide
sequence is a RNA sequence and has at least 50%, 60%, 70%, 80%,
85%, 90%, 94%, 97%, 99%, 100% homology (or, advantageously,
identity) with the sequence from position 31 to position 1411 (SEQ
ID NO: 3) (advantageously from position 169 to position 1411-SEQ ID
NO: 4) of the polynucleotide of FIG. 2 in the article by Anson D S,
Choo K H, Rees D J, Giannelli F, Gould K, Huddleston J A and
Brownlee G G. The gene structure of human anti-hemophilic factor
IX. The EMBO Journal 1984; 3: 1053-1060. In this case (that is,
with reference to SEQ ID NO: 3 and SEQ ID NO: 4), the position
numbers refer to the numbering reported in the aforementioned FIG.
2.
[0062] Advantageously, the RNA sequence has at least 80% homology
(or, advantageously, identity) with the sequence SEQ ID NO: 3
(advantageously, SEQ ID NO: 4). Advantageously, the RNA sequence
has at least 90% homology (or, advantageously, identity) with the
sequence SEQ ID NO: 3 (advantageously, SEQ ID NO: 4).
Advantageously, the RNA sequence has at least 95% homology (or,
advantageously, identity) with the sequence SEQ ID NO: 3
(advantageously, SEQ ID NO: 4).
[0063] The RNA sequence can be linked, at the head and/or tail, to
additional nucleotide chains that are either not translated or
translated separately.
[0064] According to some alternative embodiments, the nucleotide
sequence is a DNA sequence and comprises (in particular, consists
of) intron and exon portions, which present an overall sequence
(that is to say exon portions without gaps and linked together in
order) having at least 50%, 60%, 70%, 80%, 85%, 90%, 94%, 97%, 99%,
100% homology (or, advantageously, identity) with the overall
sequence of exon regions in the sequence (SEQ ID NO: 5) of FIG. 4
in the article by Anson D S, Choo K H, Rees D J, Giannelli F, Gould
K, Huddleston J A and Brownlee G G. The gene structure of human
anti-hemophilic factor IX. The EMBO Journal 1984;3:1053-1060.
[0065] Advantageously, the exon portions are separate from each
other and placed in order (arranged relative to each other) as are
the respective exon regions in the sequence SEQ ID NO: 5.
Advantageously, the overall sequence of the exon portions has at
least 80% homology (or, advantageously, identity) with the overall
sequence of the exon regions. Advantageously, the overall sequence
of the exon portions has at least 90% homology (or, advantageously,
identity) with the overall sequence of the exon regions.
Advantageously, the overall sequence of the exon portions has at
least 95% homology (or, advantageously, identity) with the overall
sequence of the exon regions.
[0066] According to some embodiments, the nucleotide sequence
comprises a thymine in a position corresponding to position 34099
(or in the corresponding position 32318 according to the numbering
given in SEQ ID NO: 5; or in the corresponding position 31134
according to the numbering given in the Database of mutations of
Hemophilia B (Giannelli et al., Hemophilia B: Database of point
mutations and short additions and deletions. Nucleic Acids Research
1990;18:4053-9); or a uracil in the corresponding position 11180 of
SEQ ID NO: 3 or SEQ ID NO: 4).
[0067] In other words, the aforementioned nucleotide sequence
differs from the sequence having accession number K02402 (GenBank)
by at least the fact of bearing a mutation from guanine to thymine
in position 34099 (G34099T) or in a corresponding position (for
example position 32318 according to the numbering of SEQ ID NO: 5;
or from guanine to uracil in the corresponding position 11180 of
SEQ ID NO: 3 or SEQ ID NO: 4).
[0068] In this case the nucleotide sequence codes for a leucine in
a position corresponding to position 338.
[0069] According to some embodiments, the nucleotide sequence in
the positions corresponding to 34098, 34099 and 34100, presents a
triplet chosen from the group consisting of: TTA, UUA, TTG, UUG,
CTT, CUU, CTC, CUC, CTA, CUA, CTG, CUG, GAT, GAU, GAC, CAA, CAG. In
particular, when the nucleotide sequence is a DNA sequence, the
triplet is chosen from the group consisting of TTA, TTG, CTT, CTC,
CTA, CTG, GAT, GAC, CAA, CAG.
[0070] According to some embodiments, the nucleotide sequence, in
the positions corresponding to 34098, 34099 and 34100, presents a
triplet chosen from the group consisting of: TTA, UUA, TTG, UUG,
CTT, CUU, CTC, CUC, CTA, CUA, CTG, CUG, CAA, CAG. In particular,
when the nucleotide sequence is a DNA sequence, the triplet is
chosen from the group consisting of TTA, TTG, CTT, CTC, CTA, CTG,
CAA, CAG. In these cases, the sequence codes for a leucine or a
glutamine in a position corresponding to position 338.
[0071] According to some embodiments, the nucleotide sequence, in
the positions corresponding to 34098, 34099 and 34100, presents a
triplet chosen from the group consisting of: TTA, UUA, TTG, UUG,
CTT, CUU, CTC, CUC, CTA, CUA, CTG, CUG. In particular, when the
nucleotide sequence is a DNA sequence, the triplet is chosen from
the group consisting of TTA, TTG, CTT, CTC, CTA, CTG.
Advantageously, the triplet is CTA. In these cases, the sequence
codes for a leucine in a position corresponding to position
338.
[0072] According to some embodiments, the nucleotide sequence, in
the positions corresponding to 34098, 34099 and 34100, presents a
triplet chosen from the group consisting of: CAA, CAG. In these
cases, the sequence codes for a glutamine in a position
corresponding to position 338. Advantageously, the triplet is CAA.
To obtain the CAA triplet, an adenine is inserted in place of the
guanine in position 34099.
[0073] According to some embodiments, the nucleotide sequence, in
the positions corresponding to 34098, 34099 and 34100, presents a
triplet chosen from the group consisting of: GAT, GAU, GAC, CAA,
CAG. In particular, when the nucleotide sequence is a DNA sequence,
the triplet is chosen from the group consisting of GAT, GAC, CAA,
CAG. In these cases, the sequence codes for an aspartic acid or a
glutamine in a position corresponding to position 338.
[0074] According to some embodiments, the nucleotide sequence, in
the positions corresponding to 34098, 34099 and 34100, presents a
triplet chosen from the group consisting of: GAT, GAU, GAC. In
particular, when the nucleotide sequence is a DNA sequence, the
triplet is chosen from the group consisting of GAT, GAC. In these
cases, the sequence codes for an aspartic acid in a position
corresponding to position 338. Advantageously, the triplet is GAT.
To obtain the GAT triplet, a guanine is inserted in place of the
adenine in position 34098, an adenine in place of the guanine in
position 34099 and a thymine in place of the adenine in position
34100.
[0075] The aforesaid homology (or identity) percentages are
calculated without considering the specific mutated positions
indicated. In other words, for example, the sequence SEQ ID NO: 2
modified with a leucine in position 338 is considered as having
100% homology (and identity) with the unmodified sequence SEQ ID
NO: 2.
[0076] According to a third aspect of the present invention, a
nucleic acid is provided which comprises a nucleotide sequence
according to the second aspect of the present invention.
[0077] According to some embodiments, the nucleic acid comprises a
promoter in operational linkage with the nucleotide sequence.
[0078] According to a fourth aspect of the present invention, a
vector is provided comprising a nucleic acid as aforedefined in
relation to the third aspect of the present invention. In
particular, the vector comprises a nucleotide sequence according to
the second aspect of the present invention.
[0079] According to some embodiments, the vector is chosen from: a
prokaryote vector, a eukaryote vector or a viral vector.
[0080] Advantageously, the vector is a viral vector. In particular,
the vector is chosen from: an adenovirus, a retrovirus, a
herpesvirus, a lentivirus, a poxvirus, a cytomegalovirus.
[0081] According to a fifth aspect of the present invention, a
method for the production of a modified FIX polypeptide is
provided, whereby the modified FIX polypeptide is expressed by
means of a nucleic acid according to the third aspect of the
present invention.
[0082] According to some embodiments the method comprises the steps
of: introducing a vector of the fourth aspect of the present
invention into a cell; and culturing the cell such that the FIX
polypeptide is expressed.
[0083] Alternatively, the modified FIX polypeptide can be produced
by a host animal or in vitro from the aforementioned nucleotide
sequence.
[0084] According to a particular aspect of the present invention,
the method comprises the steps of: introducing the nucleotide
sequence of the second aspect of the present invention into a
cell-free system; expressing the modified polypeptide in the
cell-free system.
[0085] According to a further particular aspect of the present
invention, the method allows the modified FIX polypeptide to be
expressed in a transgenic animal comprising a nucleic acid in
accordance with the third aspect of the present invention (in
particular, the nucleotide sequence of the second aspect of the
present invention). Useful hosts for expression of the modified FIX
polypeptide include: E. coil, yeasts, plants, insect cells,
mammalian cells (Pham et al. (2003) Biotechnol. Bioeng. 84:332-42;
Bon et al. (1998) Semin Hematol. 35 (2 Suppl 2): 11-17; Wahij et
al., J. Biol. Chem. 280 (36) 31603-31607) and transgenic
animals.
[0086] The hosts can vary as to their levels of protein production
and also the types of modifications induced in the modified FIX
polypeptide subsequent to transcription. Eukaryote hosts can
include yeasts such as Saccharomyces cerevisiae and Pichia pastoris
(Skoko et al. (2003) Biotechnol. Appl. Biochem. 38 (Pt 3): 257-65),
insect cells (Muneta et al. (2003) J. Vet. Med. Sci. 65(2):
219-23), plants and cells from plants such as tobacco, rice, algae
(Mayfield et al. (2003) PNAS 100:438-442) etc. The plants are
typically modified by direct transfer of DNA and
agrobacterium-mediated transformations. Advantageously usable
vectors comprise promoter sequences and transcription termination
and control elements.
[0087] Yeasts are usually modified by replicating episomal vectors
or by a stable chromosomal integration by homologous recombination.
Advantageously, promoters are used to regulate gene expression.
Examples of promoters include GAL1, GAL7, GALS, CUP1. Proteins
produced by yeasts are usually soluble; alternatively, proteins
expressed in yeasts can be secreted.
[0088] Expression in eukaryotic hosts also includes production in
animals, for example in serum, milk and eggs. Transgenic animals
for the production of FIX polypeptides are known (for example
US2002/0166130 and US2004/0133930) and can be adapted for producing
the modified FIX polypeptide as aforedefined.
[0089] Prokaryote cells in particular E. coli can be advantageously
utilized to produce large quantities of modified FIX polypeptide as
aforedefined (Platis et al. (2003) Protein Exp. Purif.
31(2):222-30; Khalizzadeh et al. (2004) J. Ind. Microbial.
Biotechnol. 31(2): 63-69).
[0090] The vectors used with E. coli advantageously contain
promoters able to induce high levels of protein expression and to
express proteins that show some toxicity towards the host cells.
Examples of promoters are T7 and SP6 RNA.
[0091] Reducing agents such as .beta.-mercaptoethanol can be
utilized to solubilise polypeptides which may precipitate in the
cytoplasmic environment of E. coli.
[0092] According to a sixth aspect of the present invention, a
modified FIX polypeptide is also provided in accordance with the
first aspect of the present invention, for use as a medicament.
[0093] The modified FIX polypeptide can be used for disease
treatments either alone or in combination with other active
compounds.
[0094] The modified FIX polypeptide is useful for treating
coagulopathies (congenital or acquired), haematological diseases
(congenital or acquired), haemorrhagic disorders (such as
haemorrhagic gastritis and/or uterine bleeding), other
cardiovascular diseases.
[0095] According to some embodiments, the modified FIX polypeptide
is provided for the treatment of at least one coagulopathy.
[0096] According to some embodiments, the modified FIX polypeptide
is provided for the treatment of haematological diseases.
[0097] According to some embodiments, the modified FIX polypeptide
is provided for the treatment of haemorrhagic disorders.
[0098] According to some embodiments, the modified FIX polypeptide
is administered to patients periodically for relatively long time
periods or before, during and/or after surgical procedures to
reduce and/or prevent haemorrhages.
[0099] The use of modified FIX polypeptide for the treatment of
coagulopathies is particularly effective.
[0100] Advantageously, modified FIX polypeptide is used for the
treatment of haemophilia, and in particular haemophilia A and
haemophilia B.
[0101] According to advantageous embodiments, the modified FIX
polypeptide is provided for treating haemophilia B, and
advantageously severe and/or moderate haemophilia B.
[0102] Advantageously, modified FIX polypeptide is used for the
treatment of mammals, in particular human patients.
[0103] According to a seventh and an eighth aspect of the present
invention, the following are provided: use of the modified FIX
polypeptide in accordance with the first aspect of the present
invention for preparing a drug (pharmaceutical preparation)
advantageously for treating a coagulopathy; and a pharmaceutical
preparation comprising the modified FIX polypeptide and,
advantageously, at least one pharmaceutically acceptable
excipient.
[0104] According to some embodiments, the pharmaceutical
preparation is for the treatment of a pathology chosen from the
group consisting of: coagulopathies (congenital or acquired),
haematological diseases (congenital or acquired), haemorrhagic
disorders (such as haemorrhagic gastritis and/or uterine bleeding),
haemophilia (haemophilia A or haemophilia B). According to specific
embodiments, the pharmaceutical preparation is for treating a
coagulopathy. According to specific embodiments, the pharmaceutical
preparation is for treating haemophilia.
[0105] According to a further aspect of the present invention, a
method is provided for treating at least one coagulopathy, this
method allowing the administration of an effective quantity of a
modified FIX polypeptide as aforedefined.
[0106] The modified FIX polypeptide can be administered as a pure
compound, but is advantageously presented in the form of a
pharmaceutical preparation. Non-limiting examples of pharmaceutical
preparations if needed for this purpose are explained below.
[0107] The modified FIX polypeptide can be formulated for oral,
parenteral or rectal administration, or in forms suited to
administrations by inhalation or insufflation (either via the mouth
or nose). Formulations for oral or parenteral administration are
advantageous.
[0108] For oral administrations, the pharmaceutical preparations
are in the form of, for example, tablets or capsules prepared by
known methods with pharmaceutically acceptable excipients such as
binders (for example pregelatinized maize starch,
polyvinylpyrrolidone, or methyl cellulose); fillers (for example
lactose, microcrystalline cellulose or calcium hydrogen phosphate);
additives (for example magnesium stearate, talc, silica);
disintegrants (for example potato starch); and/or lubricants (for
example sodium lauryl sulphate). The tablets can be coated using
known methods. Liquid preparations for oral administration have the
form, for example, of solutions, syrups or suspensions, or can be
in the form of a dry product that can be dissolved in water or
another liquid prior to use. Said preparations are prepared by
known methods with pharmaceutically acceptable additives such as
suspending agents (for example sorbitol, cellulose derivatives,
edible hydrogenated fats); emulsifying agents (for example lecithin
or acacia); non-aqueous liquids (for example almond oil, oily
esters, ethyl alcohol or fractionated vegetable oils); and/or
preservatives (for example methyl or propyl-hydroxybenzoates,
sorbic acid or ascorbic acid). The preparations can also contain,
in appropriate cases, buffering salts, colouring agents, flavouring
agents and/or sweeteners.
[0109] Preparations for oral administration are formulated in a
known manner, in order to provide a controlled release of the
active compound.
[0110] The modified FIX polypeptide is formulated, in a known
manner, for parenteral administration, by injection or continuous
administration. Formulations for injection are, advantageously, in
the form of dosage units, for example in ampoules or multi-dose
containers containing preservatives. The composition can be in the
form of a suspension, in aqueous or oily liquids, and can contain
elements of the formulation as dispersing and stabilizing agents.
Alternatively, the active compound can be in powder form to be
dissolved just before use in a liquid as needed. such as sterile
water.
[0111] The modified FIX polypeptide can be formulated for rectal
administration as suppositories or enemas, for example, containing
suppository excipients of known type such as cocoa butter or other
glycerides.
[0112] The modified FIX polypeptide is also formulated, in a known
manner, in extended release compositions. These extended release
compositions are, for example, administered by means of an implant
(for example subcutaneous or intramuscular) or an intramuscular
injection. Therefore, for example, the modified FIX polypeptide is
formulated with suitable polymer or hydrophobic materials (such as
an emulsion or an oil) or ion exchange resins, or relatively poorly
soluble derivatives, such as relatively poorly soluble salts.
[0113] For intranasal administration, the modified FIX polypeptide
is formulated by administrations via a (known) device, such as in a
powder with a suitable vehicle. The dosages of the modified FIX
polypeptide will depend on the patient age and condition, and so
the precise dosage will have to be decided each time by the
physician. The dosage will also depend on the mode of
administration and the particular compound selected. Usable doses
can be for example comprised between 0.1 mg/kg and 400 mg/kg body
weight per day.
[0114] According to a further aspect of the present invention, the
nucleotide sequence is provided in accordance with the second
aspect of the present invention for use as a medicament
(advantageously for treating a coagulopathy).
[0115] The nucleotide sequence can be used for treating a pathology
either alone or in combination with other active compounds.
[0116] The nucleotide sequence is useful for treating the
pathologies of the sixth aspect of the present invention.
[0117] According to particular aspects of the present invention,
the following are provided: the use of the aforementioned
nucleotide sequence for preparing a drug advantageously for
treating a coagulopathy; and a pharmaceutical preparation
containing the nucleotide sequence.
[0118] Instead of administering the modified FIX polypeptide it is
possible to administer the nucleotide sequence which encodes
it.
[0119] The nucleotide sequence can be inserted into cells or
tissues by means of any known method. The nucleotide sequence can
be incorporated into a vector for subsequent manipulations.
[0120] For example, certain cells could be engineered so as to
express the modified FIX polypeptide, by integrating the
aforementioned nucleotide sequence into a genomic location
operatively linked with the promoter sequences. Said cells can be
administered to a patient locally or systemically.
[0121] Usable viral vectors include poxvirus, herpesvirus,
retrovirus, adenovirus, adeno-associated virus and other viruses
suitable for gene therapy.
[0122] The vectors can remain as episomal or can be integrated into
the chromosomes of the treated individual. Adenovirus serotypes are
commercially available from the American Type Culture Collection
(ATCC, Rockville).
[0123] The viral vectors, in particular adenovirus, are used ex
vivo; for example, cells are isolated from a patient and transduced
with an adenovirus expressing the modified FIX polypeptide. After a
suitable period of culturing, the transduced cells are administered
to the patient locally or systemically.
[0124] Alternatively, the viruses, in particular adenoviruses,
which express the modified FIX polypeptide are isolated and
formulated with a pharmaceutically acceptable excipient and
administered to the patient. Typically, the adenoviruses are
administered at doses of 1 to 1014 particles per kilogram of
patient weight, generally from 106 to 1012 particles per kilogram
of patient weight.
[0125] Additional examples of cell types for the expression and
release of the modified FIX polypeptide are fibroblasts and
endothelial cells (Palmer et al. (1989) Blood 73:483-445; Yao et al
(1991) PNAS 88:8101-8105).
[0126] A vehicle which presents the aforementioned nucleotide
sequence can be formulated in a similar manner to that described
above for the modified FIX polypeptide.
[0127] The nucleotide sequence and/or drugs and/or vehicles
presenting said nucleotide sequence can be used for treating the
pathologies referred to above in relation to the modified FIX
peptide.
[0128] Advantageously, the aforementioned nucleotide sequence is
used for treating mammals, in particular human patients.
[0129] According to a further aspect of the present invention, a
method is provided for detecting the protein of the first aspect of
the present invention and/or the nucleotide sequence of the second
aspect of the present invention.
[0130] Usable methods are those known in the state of the art, and
can be adapted to those polymorphism under study to include for
example immunoenzymatic assays, coagulation protein activity tests
(including FIX activity), coagulometric and chromogenic tests.
[0131] According to some embodiments, the method comprises a step
of amplifying by PCR part of a nucleic acid molecule (in which it
is required to verify the presence of the nucleotide sequence of
the second aspect of the present invention).
[0132] Advantageously, the amplification step is preceded by a step
of purifying, in particular isolating, the nucleic acid
molecule.
[0133] Advantageously the amplification step is followed by a
sequencing step.
[0134] By way of example, the methods of examples 2 and 3 below can
be followed to detect the aforesaid nucleotide sequence.
[0135] The method for detecting the protein and/or nucleotide
sequence can be used to assist in the identification of those
individuals who display a high tendency to develop blood diseases
such as thrombosis.
[0136] Further characteristics of the present invention will ensue
from the following description of some examples which are merely
illustrative and non-limiting.
EXAMPLE 1
Routine Laboratory Tests Carried out on the Proband
[0137] Routine laboratory coagulation tests were carried out with
regard to thrombophilia screening on an individual (defined as the
Proband) exhibiting episodes of deep vein thrombosis but no other
health problems.
[0138] In particular, the following were carried out: prothrombin
time, partial thromboplastin time, factor IX levels, factor VIII
and Xl levels, antithrombin levels (activity and antigen), protein
C levels (coagulometric and chromogenic activity, antigen), protein
S levels (total antigen, free antigen and activity), activated
protein C resistance, DNA analysis for factor V Leiden, DNA
analysis for the prothrombin variant G20210A, antiphospholipid
antibodies, plasminogen, fibrinolysis tests. The coagulation tests
carried out on the Proband were all found to be within normal
limits except for FIX activity (see example 4 below).
EXAMPLE 2
Isolation of Mutant FIX from Plasma and from the Cell Culture
medium.
[0139] Isolation of FIX from plasma or from culture medium was
achieved by means of the immunoaffinity column technique, using a
resin (sepharose 4B) to which the anti-FIX monoclonal antibody
AHIX-5041 [Haematologic Technologies, Inc. (Essex Junction, Vt.,
USA)] was covalently bound (3.5 mg of monoclonal antibody per 3 ml
of sepharose resin). Briefly, the column was equilibrated with
buffer containing 20 mM Tris, 150 mM NaCI, 1 mM benzamidine
(mM=millimolar). Starting from the plasma, vitamin K-dependent
factors were precipitated by adding barium chloride. After
centrifugation, the sediment was resuspended in a solution
containing 0.2 M EDTA. The preparation thus obtained was
extensively dialyzed (2 times, for at least 2 hours) in a solution
containing 20 mM Tris, 150 mM NaCI. After dialysis, the preparation
was permitted to pass through the column at a rate of 0.5 ml/min.
After extensive column washing (10 column volumes) with Tris/NaCI
buffer, elution was carried out using a solution of acidic glycine
(pH 2.45). The eluate pH was immediately neutralized by adding 2 M
Tris at pH 7.5. The eluate fractions containing protein (tested by
the Bradford protein assay) were pooled and dialyzed against a
Tris-NaCI solution, the FIX was then concentrated through a 2000
microcolumn of fast-flow sepharose Q (ion exchange). The purity of
the preparation was evaluated by applying the silver staining
technique on the SDS-PAGE gel.
EXAMPLE 3
Genetic Study of FIX
[0140] PCR amplification and direct sequencing of the exons and
splice sites of the Proband FIX gene were carried out using
standardized techniques and primers as reported in the literature
(From: Methods in Molecular Medicine, Vol 31: Hemostasis and
Thrombosis Protocols. Edited by D. J. Perry and K. J. Pasi. Humana
Press Inc. Totowa, N.J. Chapter 16: Hemophilia B mutational
analysis. By Peter Green). Briefly, amplification was carried out
by using intron primer pairs flanking each of the eight exons of
the FIX gene. The sequencing was undertaken with an ABI PRISM 310
sequencer (Perkin Elmer, Foster City, Calif.) using the ABI PRISM
BigDye Terminator kit for cycle sequencing reactions. The sequence
data were analyzed using the Sequencing Analysis 3.0 programme
(Perkin Elmer, Calif.). The sequence obtained was compared with the
FIX sequence reported on the GenBank database (accession number:
K02402).
[0141] Analysis of the nucleotide sequence of the Proband FIX gene
has documented a single mutation in exon VIII of the FIX gene
compared to the normal sequence. The patient was found to be a
carrier for a mutation from G to T at position 34099 of the FIX
gene (normal sequence of the FIX gene, Gene bank accession number:
K02402) (or in the corresponding position 31134 according to the
numbering given in the Database of mutations of Hemophilia B
(Giannelli et al., Hemophilia B: Database of point mutations and
short additions and deletions. Nucleic Acids Research
1990;18:4053-9) able to change codon 338 from Arginine to Leucine.
Therefore the FIX molecule present in the Proband's plasma (mutated
FIX) differs from the normal FIX molecule only by the presence of
the amino acid substitution in position 338 where there is a
Leucine instead of Arginine.
EXAMPLE 4
In Vitro Mutagenesis, Expression and Purification of Recombinant
FIX Containing the Leu 338 Mutation
[0142] Site-specific mutagenesis was carried out according to
standard techniques described by Kunkel (Kunkel TA. Rapid and
efficient site-specific mutagenesis without phenotypic selection;
Proc Nati Acad Sci, USA 1985, 82:488-492). Sequencing of the cDNA
was carried out for assurance that the mutation was correct and
that any new mutations had not been introduced. Expression of the
recombinant FIX was obtained using "human embryonic kidney cell
line 293" and the methods already reported in the literature (Chang
J L, Jin J P, Lollar P, et al. Changing residue 338 in human factor
IX from arginine to alanine causes an increase in catalytic
activity. J. Biol. Chem. 1998;273:12089-12094). The recombinant FIX
was isolated from the supernatant (culture medium) by means of an
immunoaffinity column, as aforedescribed. Briefly, the supernatant
of the cell culture was collected every 24 hours for 10 days and
conserved at -20.sup.2C. For the purification the supernatant was
thawed out and benzamidine and EDTA were added to a final
concentration of 5 milliMoles and 4 milliMoles respectively. After
filtration through a Millipore filter, the supernatant was
incubated with fast-flow Sepharose Q resin for 12 hours at
4.degree. C. The resin was then re-equilibrated in Tris, NaCI and
benzamidine buffer and loaded onto the column. Elution was
undertaken with a 0-60 nM calcium gradient. The eluate was then
dialyzed in a Tris-NaCI buffer. The preparation was then applied to
the immunoaffinity column following the method described in example
2 (in the "in vitro" expression of the recombinant protein).
Starting from the culture medium, the procedure was the same as for
the plasma, except for the precipitation procedure using BaCI. The
culture medium was centrifuged at 4000 g for 20 minutes then
subjected to dialysis in Tris-NaCI and loaded onto the
immunoaffinity column at a rate of 0.5 ml/min.
[0143] The remaining steps were the same as those taken for the
plasma.
[0144] The FIX with the G34099T gene mutation resulting in the
338Leu amino acid substitution, was obtained by in vitro
mutagenesis and expression techniques. The level of expression in
cell culture was found to be similar to that obtainable with
non-mutated recombinant FIX (normal molecule). Specifically, the
expression level of the non-mutated recombinant FIX was between 750
and 880 ng/ml while for the recombinant factor IX with the gene
mutation G340991 resulting in the 338Leu amino acid substitution,
the level was between 590 and 629 ng/ml.
EXAMPLE 5
Functional Assay of FIX
[0145] The functional assay of FIX was carried out on the Proband's
plasma with a coagulometric test using Actin (Dade Behring,
Marburg, Germany) and FIX deficient plasma (Dade Behring, Marburg,
Germany). Briefly for the coagulometric test a variant of the
partial thromboplastin time (PTT) was used in a system containing
FIX deficient plasma. After adding the calcium chloride the
clotting time was measured in seconds. This clotting time was
compared to those of a calibration curve obtained by serial
dilutions of a pool of normal plasma as reference containing FIX at
a quantity of 5 .mu.g/ml (i.e. 100%), and the FIX percentage
present in the sample being calculated on 100% of the normal plasma
pool (according to common standardized methods).
[0146] The normal range for the test had been previously obtained
by analyzing, using the same method, 100 healthy individuals of
both sexes, aged between 20 and 70 years.
[0147] The activity levels of FIX in the Proband were found to be
equal to 776% (normal range in 100 healthy individuals,
80-120%).
EXAMPLE 6
Assay of FIX Antigen
[0148] The FIX antigen was determined with the ELISA test using a
first anti-FIX monoclonal antibody (Affinity Biologicals, Ontario,
Canada) coated (bound) onto the plate for the capture and a second
monoclonal antibody labelled with Horseradish peroxidase (HRP)
(Affinity Biologicals, Canada) for the detection of FIX. The
reference curve was constructed by diluting a pool of normal plasma
from 1:100 to 1:3200 in a buffer for the samples, according to
standardized procedures. Briefly, the first antibody was bound to
the plate after dilution in sodium bicarbonate buffer at basic pH
(pH=9.0) at a final concentration of 4 .mu.g/ml. After extensive
washing of the plate with Tris-NaCl-Tween20 buffer, the samples,
diluted 1:100 and 1:200 in the same buffer, were loaded into the
wells and incubated at ambient temperature for 2 hours. After
removal of the samples from the wells and extensive washing with
the buffer, 100 .mu.l of a solution containing the second antibody
conjugated with HRP were added to each of the wells and incubated
at ambient temperature for two hours. After further washes, 100
.mu.l of a solution containing tetramethylbenzidine (TMB) were
added and the developed color was measured by spectrophotometer
with a 450 nanometer filter. The level of FIX antigen was
calculated using the reference curve and expressed as a percentage
of the pool of normal plasma. The normal test range was previously
obtained using the same method by analyzing 100 healthy individuals
of both sexes, aged between 20 and 70 years.
[0149] FIX antigen levels were found to be equal to 92% (normal
range 80-120%). This result (combined with that obtained in example
5) was compatible with the presence of normal quantities of a
synthesized circulating FIX, but with its procoagulant function
being around 8-9 times greater than the normal FIX molecule.
EXAMPLE 7
Activity and Antigen Levels of FIX after Reconstitution of a FIX
Deficient Plasma with an FIX Extracted from the Proband's Plasma
and with Recombinant FIX
[0150] After isolating FIX from the Proband's plasma, this FIX was
used for reconstituting a FIX deficient plasma (Dade-Behring,
Milan, Italy) with a final FIX concentration of 5 .mu.g/ml (equal
to 100% of normal). The measurements of FIX activity and antigen in
the thus reconstituted plasma were 740% and 95% respectively, these
being hence comparable with those of the Proband's plasma.
[0151] For assaying the activity of the recombinant FIX obtained in
accordance with example 4, the same system was used after
recomposition of a FIX deficient plasma with a quantity of mutated
recombinant FIX (rFIX 338Leu) such as to restore the normal FIX
concentration in normal human plasma, i.e. 5 .mu.g/ml
(corresponding to 100% of normal) (.mu.g=micrograms). The
measurements of recombinant factor IX activity and antigens were
780% and 90% respectively, these being hence comparable with those
of the Proband's plasma. This indicates that the recombinant
protein thus obtained, containing the amino acid substitution also
present in factor IX of the Proband, has a biological activity at
least 8-9 times greater than normal factor IX.
EXAMPLE 8
SDS-PAGE and Immunoblotting of FIX
[0152] The SDS-PAGE and immunoblotting (Western blot) of the FIX
was carried out on a 5-15% linear gradient gel according to
standard procedures. Briefly, the samples containing normal FIX or
recombinant FIX were loaded into the polyacrylamide gel wells and
subjected to electrophoresis.
[0153] The FIX was then subjected to transblotting on a
polyvinylidene fluoride (PVDF) membrane using a semidry apparatus
(Novablot, GE-Healthcare, Milan, Italy).
[0154] The FIX was detected on the PVDF membrane after
transblotting using an anti-FIX monoclonal antibody conjugated to
HRP (Affinity Biologicals, Ontario, Canada).
[0155] FIG. 1 shows that the FIX isolated from the Proband, the
338Leu recombinant FIX and the normal FIX exhibit the same
electrophoretic mobility and the same immunoblot pattern (in FIG.
1, 1 indicates molecular weight markers, 2 indicates normal FIX, 3
indicates natural modified FIX, 4 indicates the modified
recombinant FIX).
[0156] Therefore no significant differences (neither quantitative
nor qualitative) between normal human FIX, 338 Leu natural mutant
human FIX and 338Leu recombinant FIX were found using this
technique.
[0157] From the aforedescribed, it is clear that the presence of a
leucine in a position corresponding to position 338 surprisingly
increases the activity of FIX polypeptide by almost eight
times.
[0158] The present invention proves to be a particular improvement
on the state of the art as it provides a modified FIX polypeptide
which in vivo in man does not cause any side effects other than an
increased coagulation activity.
EXAMPLE 9
In Vitro Mutagenesis, Expression and Purification of the
Recombinant FIX Containing the 338 Asp Mutation (338 Aspartic Acid,
338D)
[0159] The site-specific mutagenesis was carried out according to
standard techniques described by Kunkel (Kunkel TA. Rapid and
efficient site-specific mutagenesis without phenotypic selection;
Proc Nati Acad Sci USA 1985, 82: 488-492) by inserting a guanine in
place of cytosine in position 34098, and an alanine in place of
guanine in position 34099 and a thymine in place of alanine in
position 34100 (the mutagenesis was also repeated by inserting a
guanine in place of cytosine in position 34098, an adenine in place
of guanine in position 34099 and a guanine in place of adenine in
position 34100).
[0160] Sequencing of the cDNA was carried out for assurance that
the mutation was correct and that any new mutations had not been
introduced. Expression of the recombinant FIX was obtained using
"human embryonic kidney cell line 293" and the methods already
reported in the literature (Chang J L, Jin J P, Lollar P, et al.
Changing residue 338 in human factor IX from arginine to alanine
causes an increase in catalytic activity. J. Biol. Chem.
1998;273:12089-12094). The recombinant FIX was isolated from the
supernatant (culture medium) by means of an immunoaffinity column,
as aforedescribed. Briefly, the supernatant of the cell culture was
collected every 24 hours for 10 days and conserved at -20.degree.
C. For the purification the supernatant was thawed out and
benzamidine and EDTA were added to a final concentration of 5
milliMoles and 4 milliMoles respectively. After filtration through
a Millipore filter, the supernatant was incubated with fast-flow
Sepharose Q resin for 12 hours at 4.degree. C. The resin was then
re-equilibrated in Tris, NaCI and benzamidine buffer and loaded
onto the column. Elution was undertaken with a 0-60 nM calcium
gradient. The eluate was then dialyzed in a Tris-NaCI buffer. The
preparation was applied to the immunoaffinity column following the
method described in example 2 (in the "in vitro" expression of the
recombinant protein). Starting from the culture medium, the
procedure was the same as for the plasma, except for the
precipitation procedure using BaCI. The culture medium was
centrifuged at 4000 g for 20 minutes then subjected to dialysis in
Tris-NaCI and loaded onto the immunoaffinity column at a rate of
0.5 ml/min. The remaining steps were the same as those taken for
the plasma.
[0161] The FIX with the amino acid substitution 338Asp was obtained
by in vitro mutagenesis and expression techniques. The level of
expression in cell culture was found to be similar to that
obtainable with non-mutated recombinant FIX (normal molecule).
Specifically, the expression level of the non-mutated recombinant
FIX was between 750 and 880 ng/ml while for the recombinant factor
IX with the 338Asp amino acid substitution, the level was between
650 and 740 ng/ml.
EXAMPLE 10
Activity and Antigen Levels of FIX after Reconstitution of a FIX
Deficient Plasma with Recombinant FIX with 338Asp Mutation
[0162] For the assay of the activity of recombinant FIX obtained in
accordance with example 9, the same system was used after
recomposition of a FIX deficient plasma with a quantity of mutated
recombinant FIX (rFIX 338Asp) such as to restore the normal
concentration of FIX in normal human plasma, i.e. 5 .mu.g/ml
(corresponding to 100% of normal) (.mu.g=micrograms). Measurements
of recombinant factor IX activity and antigens were 460% and 98%
respectively. This indicates that the recombinant protein thus
obtained (FIX 338 Asp), has a biological activity at least 5 times
greater than normal factor IX.
EXAMPLE 11
SDS-PAGE and Immunoblotting of FIX
[0163] The SDS-PAGE and immunoblotting (Western blot) of the FIX
was carried out on a 5-15% linear gradient gel according to
standard procedures. Briefly, the samples containing normal FIX or
recombinant FIX were loaded into the polyacrylamide gel wells and
subjected to electrophoresis.
[0164] The FIX was then subjected to transblotting on a
polyvinylidene fluoride (PVDF) membrane using a semidry apparatus
(Novablot, GE-Healthcare. Milan, Italy).
[0165] The FIX was detected on the PVDF membrane after
transblotting using an anti-FIX monoclonal antibody conjugated to
HRP (Affinity Biologicals, Ontario, Canada).
[0166] The 338Asp recombinant FIX and the normal FIX exhibit the
same electrophoretic mobility and the same immunoblot pattern.
Therefore no significant differences (neither quantitative nor
qualitative) between normal human FIX and 338Asp recombinant FIX
were found using this technique.
[0167] From the aforedescribed, it is clear that the presence of an
Aspartic acid in a position corresponding to position 338
surprisingly increases the activity of FIX polypeptide by almost
eight times.
[0168] The present invention proves to be a particular improvement
on the state of the art as it provides a modified FIX polypeptide
which in vivo in man does not cause any side effects other than an
increased coagulation activity.
EXAMPLE 12
In Vitro Mutagenesis, Expression and Purification of Recombinant
FIX Containing the 338GIn Mutation (338 Glutamine, 3380)
[0169] Site-specific mutagenesis was carried out according to
standard techniques described by Kunkel (Kunkel TA. Rapid and
efficient site-specific mutagenesis without phenotypic selection;
Proc Nati Acad Sci USA 1985, 82: 488-492) by inserting an adenine
in place of guanine in position 34099 (the mutagenesis was also
repeated by inserting an adenine in place of guanine in position
34099, and a guanine in place of adenine in position 34100).
Sequencing of the cDNA was carried out for assurance that the
mutation was correct and that any new mutations had not been
introduced. Expression of the recombinant FIX was obtained using
"human embryonic kidney cell line 293" and the methods already
reported in the literature (Chang J L, Jin J P, Lollar P, et al.
Changing residue 338 in human factor IX from arginine to alanine
causes an increase in catalytic activity. J. Biol. Chem.
1998;273:12089-12094). The recombinant FIX was isolated from the
supernatant (culture medium) by means of an immunoaffinity column,
as aforedescribed. Briefly, the supernatant of the cell culture was
collected every 24 hours for 10 days and conserved at -20.degree.
C. For the purification the supernatant was thawed out and
benzamidine and EDTA were added to a final concentration of 5
milliMoles and 4 milliMoles respectively. After filtration through
a Millipore filter, the supernatant was incubated with fast-flow
Sepharose 0 resin for 12 hours at 4.degree. C. . The resin was then
re-equilibrated in Tris, NaCI and benzamidine buffer and loaded
onto the column. Elution was undertaken with a 0-60 nM calcium
gradient. The eluate was then dialyzed in a Tris-NaCI buffer. The
preparation was then applied to the immunoaffinity column following
the method described in example 2 (in the "in vitro" expression of
the recombinant protein). Starting from the culture medium, the
procedure was the same as for the plasma, except for the
precipitation procedure using BaCI. The culture medium was
centrifuged at 4000 g for 20 minutes then subjected to dialysis in
Tris-NaCI and loaded onto the immunoaffinity column at a rate of
0.5 ml/min. The remaining steps were the same as those taken for
the plasma.
[0170] The FIX with the amino acid substitution 338Gin was obtained
by in vitro mutagenesis and expression techniques. The level of
expression in cell culture was found to be similar to that
obtainable with non-mutated recombinant FIX (normal molecule).
Specifically, the expression level of the non-mutated recombinant
FIX was between 750 and 880 ng/ml while for the recombinant factor
IX with the 338GIn amino acid substitution, the level was between
600 and 720 ng/ml.
EXAMPLE 13
Levels of Activity and Antigen of the FIX after Reconstitution of a
FIX Deficient Plasma with Recombinant FIX with 338GIn Mutation
[0171] For the assay of the activity of recombinant FIX obtained in
accordance with example 12, the same system was used after
recomposition of a FIX deficient plasma with a quantity of mutated
recombinant FIX (rFIX 338GIn) such as to restore the normal
concentration of FIX in normal human plasma, i.e. 5 .mu.g/ml
(corresponding to 100% of normal)=micrograms). Measurements of
recombinant factor IX activity and antigens were 1360% and 99%
respectively. This indicates that the recombinant protein thus
obtained (FIX 338 Gin) has a biological activity at least 13 times
greater than normal factor IX.
EXAMPLE 14
SDS-PAGE and Immunoblotting of FIX
[0172] The SDS-PAGE and immunoblotting (Western blot) of the FIX
was carried out on a 5-15% linear gradient gel according to
standard procedures. Briefly, the samples containing normal FIX or
recombinant FIX were loaded into polyacrylamide gel wells and
subjected to electrophoresis.
[0173] The FIX was then subjected to transblotting on a
polyvinylidene fluoride (PVDF) membrane using a semidry apparatus
(Novablot, GE-Healthcare, Milan, Italy).
[0174] The FIX was detected on the PVDF membrane after
transblotting using an anti-FIX monoclonal antibody conjugated to
HRP (Affinity Biologicals, Ontario, Canada).
[0175] The 338GIn recombinant FIX and the normal FIX exhibited the
same electrophoretic mobility and the same immunoblot pattern.
Therefore no significant differences (neither quantitative nor
qualitative) between normal human FIX and 338GIn recombinant FIX
were found using this technique.
[0176] From the aforedescribed, it is clear that the presence of a
glutamine in a position corresponding to position 338 surprisingly
increases the activity of FIX polypeptide by almost thirteen
times.
[0177] The present invention proves to be a particular improvement
on the state of the art as it provides a modified FIX polypeptide
which in vivo in humans does not cause any side effects other than
an increased coagulation activity.
[0178] Therefore evidence is provided that: [0179] 1) it has been
discovered a naturally occurring FIX mutant (arginine 338 leucine)
with a 8-9 fold increased functional activity as compared to FIX
wild-type; [0180] 2) recombinant modified FIX polypeptides (not
known before) with 5 folds (FIX arginine 338 aspartic acid), 8 to 9
folds (FIX arginine 338 leucine), 13 folds (FIX arginine 338
glutamine) increased functional (procoagulant) activity,
respectively, as compared to FIX wild-type can be generated.
[0181] The use of the mutants of the invention, which show such a
specific functional activity of 5 folds or above, and in particular
8 to 9 folds as compared to FIX wild type, for medical use and in
particular for the prophylaxis and treatment of Hemophilia B
patients; said use of the mutants of the invention has never been
considered before and is part of the present invention.
[0182] The use of the mutants of the invention, which show such a
specific functional activity of 5 folds or above, and in particular
8 to 9 folds as compared to FIX wild type, for gene therapy of
Hemophilia B patients has never been considered before and is part
of the present invention.
[0183] The use of the mutants of the invention, which show a
specific functional activity of 5 folds or above, and in particular
8 to 9 folds as compared to FIX wild type, for the prophylaxis and
treatment of hemorrhagic coagulopathies other than Hemophilia B or
for gene therapy of such diseases has never been considered before
and is part of the present invention.
[0184] It has to be noted that the use of the mutants of the
invention, which show a specific functional activity of 5 folds or
above, and in particular of FIX arginine 338 leucine which shows 8
to 9 folds increased functional activity as compared to FIX wild
type, is considered optimal for the treatment of patients with
hemophilia B because of the presence of an identical naturally
occurring mutant in humans (never described before, and is part of
the present invention) which does not generate neutralizing
antibodies. In addition, the FIX functional activity levels express
by FIX arginine 338 leucine, is possibly the best option being
higher than that of FIX arginine 338 alanine (previously known and
described in WO 99/03496, with a modest increase in activity of 2
to 3 folds that of FIX wild-type) and not too high to cause
thrombotic complications in hemophilia B patients or patients with
other hemorrhagic coagulopathies.
[0185] The invention of FIX arginine 338 leucine, is also the best
choice for the use of FIX mutants in gene therapy by using viral
vectors, given the actual efficiency and yield of the method for
the treatment (partial correction) of Hemophilia B.
[0186] According to certain aspects of the present invention there
are provided polypeptides, nucleotide sequences, nucleic acids,
vectors, methods and uses in accordance with the following points.
[0187] 1. A modified FIX (factor IX) polypeptide comprising:
[0188] an amino acid chosen from the group consisting of: leucine,
cysteine, aspartic acid, glutamic acid, histidine, lysine,
asparagine, glutamine, tyrosine in a position corresponding to
position 338.
[0189] 2. A polypeptide according to claim 1 wherein the amino acid
is chosen from the group consisting of: leucine, aspartic acid.
glutamine.
[0190] 3. A polypeptide according to claim 1 wherein the amino acid
is chosen from the group consisting of: aspartic acid,
glutamine.
[0191] 4. A polypeptide according to claim 1 wherein the amino acid
is aspartic acid.
[0192] 5. A polypeptide according to claim 1 wherein the amino acid
is glutamine.
[0193] 6. A polypeptide according to claim 1 wherein the amino acid
is leucine.
[0194] 7. A polypeptide according to one of the previous points,
and having a homology of at least 70% with a peptide sequence
selected from the group consisting of: SEQ ID NO: 1 and SEQ ID NO:
2.
[0195] 8. A polypeptide according to one of the previous points,
and having a homology of at least 90% with a peptide sequence
selected from the group consisting of: SEQ ID NO: 1 and SEQ ID NO:
2.
[0196] 9. A polypeptide according to one of the previous points,
and having a percentage identity of at least 70% with a peptide
sequence selected from the group consisting of: SEQ ID NO: 1 and
SEQ ID NO: 2.
[0197] 10. A polypeptide according to one of the previous points,
and having a percentage identity of at least 90% with a peptide
sequence selected from the group consisting of: SEQ ID NO: 1 and
SEQ ID NO: 2.
[0198] 11. A polypeptide according to one of the previous points
wherein the peptide sequence is SEQ ID NO: 2.
[0199] 12. A nucleotide sequence encoding a FIX polypeptide
according to one of the previous points.
[0200] 13. A nucleotide sequence according to point 12 wherein the
nucleotide sequence is a DNA sequence and consists of intron
portions and exon portions, the exon portions having an overall
sequence with at least 70% homology relative to an overall sequence
of exon regions of a SEQ ID NO: 5 sequence.
[0201] 14. A nucleotide sequence according to point 13 wherein the
overall sequence of the exon portions has at least 90% homology
with the overall sequence of the exon regions of the SEQ ID NO: 5
sequence.
[0202] 15. A nucleotide sequence according to one of points 12 to
14 wherein the nucleotide sequence has at least 50% homology with
the sequence having the accession number K02402 (Gen Bank).
[0203] 16. A nucleotide sequence according to one of points 12 to
15, comprising in positions corresponding to 34098, 34099 and 34100
a triplet chosen from the group consisting of: TTA, UUA, TTG, UUG,
CTT, CUU, CTC, CUC, CTA, CUA, CTG, CUG, GAT, GAU, GAC, CAA,
CAG.
[0204] 17. A nucleotide sequence according to point 16, comprising
in positions corresponding to 34098, 34099 and 34100 a triplet
chosen from the group consisting of: TTA, UUA, TTG, UUG, CTT, CUU,
CTC, CUC, CIA, CUA, CTG, CUG, CAA, CAG.
[0205] 18. A nucleotide sequence according to point 16, comprising
in positions corresponding to 34098, 34099 and 34100 a triplet
chosen from the group consisting of: TTA, UUA, TTG, UUG, CTT, CUU,
CTC, CUC, CTA, CUA, CTG, CUG.
[0206] 19. A nucleotide sequence according to point 16, comprising
in positions corresponding to 34098, 34099 and 34100 a triplet
chosen from the group consisting of: CAA, CAG.
[0207] 20. A nucleotide sequence according to point 16, comprising
in positions corresponding to 34098, 34099 and 34100 a triplet
chosen from the group consisting of: GAT, GAU, GAC, CAA, CAG.
[0208] 21. A nucleotide sequence according to point 16, comprising
in positions corresponding to 34098, 34099 and 34100 a triplet
chosen from the group consisting of: GAT, GAU, GAC.
[0209] 22. A nucleotide sequence according to one of points 12 to
18, comprising a thymine in a position corresponding to position
34099.
[0210] 23. A nucleic acid comprising a nucleotide sequence
according to one of points 12 to 22.
[0211] 24. A nucleic acid according to point 23, and comprising a
promoter in operational linkage with said nucleotide sequence.
[0212] 25. A vector comprising a nucleic acid according to point 23
or 24.
[0213] 26. A method for producing a modified FIX polypeptide,
whereby the modified FIX polypeptide is expressed by means of a
nucleic acid according to point 23 or 24.
[0214] 27. A method according to point 26, comprising the steps
of:
[0215] introducing a vector of point 25 into a cell; and
[0216] culturing the cell such that the FIX polypeptide is
expressed.
[0217] 28. A modified FIX polypeptide according to one of points 1
to 11 for use as a medicament.
[0218] 29. A modified FIX polypeptide according to one of points 1
to 11 for the treatment of at least one coagulopathy.
[0219] 30. Use of a modified FIX polypeptide according to one of
points 1 to 11 for preparing a drug for the treatment of at least
one coagulopathy in a mammal.
[0220] 31. A nucleotide sequence according to one of points 12 to
22 for use as a medicament.
[0221] 32. A method for detecting the nucleotide sequence of one of
points 12 to 22.
[0222] 33. A method for detecting the modified FIX polypeptide
according to one of points 1 to 11.
[0223] 34. A method according to point 32 comprising an step of
amplification by PCR.
BIBLIOGRAPHY
[0224] Ameri A, Kurachi S, Sueishi K, Kuwahara M, Kurachi K.
Myocardial fibrosis in mice with overexpression of human blood
coagulation factor IX. Blood. 2003 Mar. 1; 101 (5):1871-3. Epub
2002 Oct 24. [0225] Chang J L, Jin J P, Lollar P, et al. Changing
residue 338 in human factor IX from arginine to alanine causes an
increase in catalytic activity. J Biol Chem 1998;273:12089-12094.
[0226] Lowe GDO. Factor IX and thrombosis. British Journal of
Haematology, 2001, 115, 507-513. [0227] Kunkel T A. Rapid and
efficient site-specific mutagenesis without phenotypic selection.
Proc Nati Acad Sci USA 1985, 82:488-492. [0228] Kurachi K, Davie E
W. Isolation and characterization of a cDNA coding for human factor
IX. Proc Natl Acad Sci USA 1982;79:6461-6464. [0229] Murphy S L,
High K A. Gene therapy for haemophilia. Br J Haematol. 2008 March;
140(5):479-87. [0230] Yoshitake S, Schach B G, Foster D C, et al.
Nucleotide Sequence of thr Gene for Human Factor IX (Antihemophilic
Factor B). Biochemistry 1985;24:3736-3750. [0231] Toomey J R,
Valocik R E, Koster P F, Gabriel M A, McVey M, Hart T K, Ohlstein E
H, Parsons A A, Barone F C. Inhibition of factor IX(a) is
protective in a rat model of thromboembolic stroke. Stroke. 2002
February; 33(2):578-85.
Sequence CWU 1
1
131461PRTHomo sapiensSIGNAL1..56SIGNAL
PEPTIDEPEPTIDE1..461SITE384..384Arg RESIDUE CORRESPONDING TO 338 OF
SEQ ID No. 2SITE384..384RESIDUE MUTATED IN THE CLAIMED INVENTION
1Met Gln Arg Val Asn Met Ile Met Ala Glu Ser Pro Gly Leu Ile Thr1 5
10 15Ile Cys Leu Leu Gly Tyr Leu Leu Ser Ala Glu Cys Thr Val Phe
Leu 20 25 30Asp His Glu Asn Ala Asn Lys Ile Leu Asn Arg Pro Lys Arg
Tyr Asn 35 40 45Ser Gly Lys Leu Glu Glu Phe Val Gln Gly Asn Leu Glu
Arg Glu Cys 50 55 60Met Glu Glu Lys Cys Ser Phe Glu Glu Ala Arg Glu
Val Phe Glu Asn65 70 75 80Thr Glu Arg Thr Thr Glu Phe Trp Lys Gln
Tyr Val Asp Gly Asp Gln 85 90 95Cys Glu Ser Asn Pro Cys Leu Asn Gly
Gly Ser Cys Lys Asp Asp Ile 100 105 110Asn Ser Tyr Glu Cys Trp Cys
Pro Phe Gly Phe Glu Gly Lys Asn Cys 115 120 125Glu Leu Asp Val Thr
Cys Asn Ile Lys Asn Gly Arg Cys Glu Gln Phe 130 135 140Cys Lys Asn
Ser Ala Asp Asn Lys Val Val Cys Ser Cys Thr Glu Gly145 150 155
160Tyr Arg Leu Ala Glu Asn Gln Lys Ser Cys Glu Pro Ala Val Pro Phe
165 170 175Pro Cys Gly Arg Val Ser Val Ser Gln Thr Ser Lys Leu Thr
Arg Ala 180 185 190Glu Ala Val Phe Pro Asp Val Asp Tyr Val Asn Ser
Thr Glu Ala Glu 195 200 205Thr Ile Leu Asp Asn Ile Thr Gln Ser Thr
Gln Ser Phe Asn Asp Phe 210 215 220Thr Arg Val Val Gly Gly Glu Asp
Ala Lys Pro Gly Gln Phe Pro Trp225 230 235 240Gln Val Val Leu Asn
Gly Lys Val Asp Ala Phe Cys Gly Gly Ser Ile 245 250 255Val Asn Glu
Lys Trp Ile Val Thr Ala Ala His Cys Val Glu Thr Gly 260 265 270Val
Lys Ile Thr Val Val Ala Gly Glu His Asn Ile Glu Glu Thr Glu 275 280
285His Thr Glu Gln Lys Arg Asn Val Ile Arg Ile Ile Pro His His Asn
290 295 300Tyr Asn Ala Ala Ile Asn Lys Tyr Asn His Asp Ile Ala Leu
Leu Glu305 310 315 320Leu Asp Glu Pro Leu Val Leu Asn Ser Tyr Val
Thr Pro Ile Cys Ile 325 330 335Ala Asp Lys Glu Tyr Thr Asn Ile Phe
Leu Lys Phe Gly Ser Gly Tyr 340 345 350Val Ser Gly Trp Gly Arg Val
Phe His Lys Gly Arg Ser Ala Leu Val 355 360 365Leu Gln Tyr Leu Arg
Val Pro Leu Val Asp Arg Ala Thr Cys Leu Arg 370 375 380Ser Thr Lys
Phe Thr Ile Tyr Asn Asn Met Phe Cys Ala Gly Phe His385 390 395
400Glu Gly Gly Arg Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro His Val
405 410 415Thr Glu Val Glu Gly Thr Ser Phe Leu Thr Gly Ile Ile Ser
Trp Gly 420 425 430Glu Glu Cys Ala Met Lys Gly Lys Tyr Gly Ile Tyr
Thr Lys Val Ser 435 440 445Arg Tyr Val Asn Trp Ile Lys Glu Lys Thr
Lys Leu Thr 450 455 4602415PRTHomo
sapiensPEPTIDE1..415SITE338..338RESIDUE CORRESPONDING TO 384 OF SEQ
ID No. 2SITE338..338RESIDUE MUTATED IN THE CLAIMED INVENTION 2Tyr
Asn Ser Gly Lys Leu Glu Glu Phe Val Gln Gly Asn Leu Glu Arg1 5 10
15Glu Cys Met Glu Glu Lys Cys Ser Phe Glu Glu Ala Arg Glu Val Phe
20 25 30Glu Asn Thr Glu Arg Thr Thr Glu Phe Trp Lys Gln Tyr Val Asp
Gly 35 40 45Asp Gln Cys Glu Ser Asn Pro Cys Leu Asn Gly Gly Ser Cys
Lys Asp 50 55 60Asp Ile Asn Ser Tyr Glu Cys Trp Cys Pro Phe Gly Phe
Glu Gly Lys65 70 75 80Asn Cys Glu Leu Asp Val Thr Cys Asn Ile Lys
Asn Gly Arg Cys Glu 85 90 95Gln Phe Cys Lys Asn Ser Ala Asp Asn Lys
Val Val Cys Ser Cys Thr 100 105 110Glu Gly Tyr Arg Leu Ala Glu Asn
Gln Lys Ser Cys Glu Pro Ala Val 115 120 125Pro Phe Pro Cys Gly Arg
Val Ser Val Ser Gln Thr Ser Lys Leu Thr 130 135 140Arg Ala Glu Thr
Val Phe Pro Asp Val Asp Tyr Val Asn Ser Thr Glu145 150 155 160Ala
Glu Thr Ile Leu Asp Asn Ile Thr Gln Ser Thr Gln Ser Phe Asn 165 170
175Asp Phe Thr Arg Val Val Gly Gly Glu Asp Ala Lys Pro Gly Gln Phe
180 185 190Pro Trp Gln Val Val Leu Asn Gly Lys Val Asp Ala Phe Cys
Gly Gly 195 200 205Ser Ile Val Asn Glu Lys Trp Ile Val Thr Ala Ala
His Cys Val Glu 210 215 220Thr Gly Val Lys Ile Thr Val Val Ala Gly
Glu His Asn Ile Glu Glu225 230 235 240Thr Glu His Thr Glu Gln Lys
Arg Asn Val Ile Arg Ile Ile Pro His 245 250 255His Asn Tyr Asn Ala
Ala Ile Asn Lys Tyr Asn His Asp Ile Ala Leu 260 265 270Leu Glu Leu
Asp Glu Pro Leu Val Leu Asn Ser Tyr Val Thr Pro Ile 275 280 285Cys
Ile Ala Asp Lys Glu Tyr Thr Asn Ile Phe Leu Lys Phe Gly Ser 290 295
300Gly Tyr Val Ser Gly Trp Gly Arg Val Phe His Lys Gly Arg Ser
Ala305 310 315 320Leu Val Leu Gln Tyr Leu Arg Val Pro Leu Val Asp
Arg Ala Thr Cys 325 330 335Leu Arg Ser Thr Lys Phe Thr Ile Tyr Asn
Asn Met Phe Cys Ala Gly 340 345 350Phe His Glu Gly Gly Arg Asp Ser
Cys Gln Gly Asp Ser Gly Gly Pro 355 360 365His Val Thr Glu Val Glu
Gly Thr Ser Phe Leu Thr Gly Ile Ile Ser 370 375 380Trp Gly Glu Glu
Cys Ala Met Lys Gly Lys Tyr Gly Ile Tyr Thr Lys385 390 395 400Val
Ser Arg Tyr Val Asn Trp Ile Lys Glu Lys Thr Lys Leu Thr 405 410
41531381RNAHomo sapiens 3ugcagcgcgu gaacaugauc auggcagaau
caccaggccu caucaccauc ugccuuuuag 60gauaucuacu cagugcugaa uguacaguuu
uucuugauca ugaaaacgcc aacaaaauuc 120ugaaucggcc aaagagguau
aauucaggua aauuggaaga guuuguucaa gggaaccuug 180agagagaaug
uauggaagaa aaguguaguu uugaagaagc acgagaaguu uuugaaaaca
240cugaaagaac aacugaauuu uggaagcagu auguugaugg agaucagugu
gaguccaauc 300cauguuuaaa uggcggcagu ugcaaggaug acauuaauuc
cuaugaaugu uggugucccu 360uuggauuuga aggaaagaac ugugaauuag
auguaacaug uaacauuaag aauggcagau 420gcgagcaguu uuguaaaaau
agugcugaua acaagguggu uugcuccugu acugagggau 480aucgacuugc
agaaaaccag aaguccugug aaccagcagu gccauuucca uguggaagag
540uuucuguuuc acaaacuucu aagcucaccc gugcugaggc uguuuuuccu
gauguggacu 600auguaaauuc uacugaagcu gaaaccauuu uggauaacau
cacucaaagc acccaaucau 660uuaaugacuu cacucggguu guugguggag
aagaugccaa accaggucaa uucccuuggc 720agguuguuuu gaaugguaaa
guugaugcau ucuguggagg cucuaucguu aaugaaaaau 780ggauuguaac
ugcugcccac uguguugaaa cugguguuaa aauuacaguu gucgcaggug
840aacauaauau ugaggagaca gaacauacag agcaaaagcg aaaugugauu
cgaauuauuc 900cucaccacaa cuacaaugca gcuauuaaua aguacaacca
ugacauugcc cuucuggaac 960uggacgaacc cuuagugcua aacagcuacg
uuacaccuau uugcauugcu gacaaggaau 1020acacgaacau cuuccucaaa
uuuggaucug gcuauguaag uggcugggga agagucuucc 1080acaaagggag
aucagcuuua guucuucagu accuuagagu uccacuuguu gaccgagcca
1140caugucuucg aucuacaaag uucaccaucu auaacaacau guucugugcu
ggcuuccaug 1200aaggagguag agauucaugu caaggagaua gugggggacc
ccauguuacu gaaguggaag 1260ggaccaguuu cuuaacugga auuauuagcu
ggggugaaga gugugcaaug aaaggcaaau 1320auggaauaua uaccaaggua
ucccgguaug ucaacuggau uaaggaaaaa acaaagcuca 1380c 138141243RNAHomo
sapiens 4auaauucagg uaaauuggaa gaguuuguuc aagggaaccu ugagagagaa
uguauggaag 60aaaaguguag uuuugaagaa gcacgagaag uuuuugaaaa cacugaaaga
acaacugaau 120uuuggaagca guauguugau ggagaucagu gugaguccaa
uccauguuua aauggcggca 180guugcaagga ugacauuaau uccuaugaau
guuggugucc cuuuggauuu gaaggaaaga 240acugugaauu agauguaaca
uguaacauua agaauggcag augcgagcag uuuuguaaaa 300auagugcuga
uaacaaggug guuugcuccu guacugaggg auaucgacuu gcagaaaacc
360agaaguccug ugaaccagca gugccauuuc cauguggaag aguuucuguu
ucacaaacuu 420cuaagcucac ccgugcugag gcuguuuuuc cugaugugga
cuauguaaau ucuacugaag 480cugaaaccau uuuggauaac aucacucaaa
gcacccaauc auuuaaugac uucacucggg 540uuguuggugg agaagaugcc
aaaccagguc aauucccuug gcagguuguu uugaauggua 600aaguugaugc
auucugugga ggcucuaucg uuaaugaaaa auggauugua acugcugccc
660acuguguuga aacugguguu aaaauuacag uugucgcagg ugaacauaau
auugaggaga 720cagaacauac agagcaaaag cgaaauguga uucgaauuau
uccucaccac aacuacaaug 780cagcuauuaa uaaguacaac caugacauug
cccuucugga acuggacgaa cccuuagugc 840uaaacagcua cguuacaccu
auuugcauug cugacaagga auacacgaac aucuuccuca 900aauuuggauc
uggcuaugua aguggcuggg gaagagucuu ccacaaaggg agaucagcuu
960uaguucuuca guaccuuaga guuccacuug uugaccgagc cacaugucuu
cgaucuacaa 1020aguucaccau cuauaacaac auguucugug cuggcuucca
ugaaggaggu agagauucau 1080gucaaggaga uaguggggga ccccauguua
cugaagugga agggaccagu uucuuaacug 1140gaauuauuag cuggggugaa
gagugugcaa ugaaaggcaa auauggaaua uauaccaagg 1200uaucccggua
ugucaacugg auuaaggaaa aaacaaagcu cac 124355280DNAHomo
sapiensCDS325..411/transl_table=1misc_feature600-601/note="intron
between positions 600-601 not shown, approx length 5400bp"
/note="position 601 corresponds to position 6000 in Anson et al
paper"CDS776..940/transl_table=1CDS1129..1152/transl_table=1misc_feature1-
200-1201/note="Intron between position 1200 and 1201, approx length
3960 bp" /note="position 1200 corresponds to position 6600 in Fig 4
of Anson et al paper, position 1201 corresponds to position 10560
in Fig 4 of Anson et
al."CDS1264..1377/transl_table=1misc_feature1440-1441/note="Intron
present between positions 1440 and 1441, approx length 7320 bp"
/note="position 1440 corresponds to 10800 in Fig 4 of Anson et al,
position 1441 corresponds to 18120 in Fig 4 of Anson et
al"CDS1582..1710/transl_table=1misc_feature1800..1801/note="Intron
between positions 1880-1801, approx length 2400 bp" /note="position
1800 corresponds to position 18480 in Fig 4 of Anson et al,
position 1801 corresponds to position 20880 of Anson et
al"CDS1881..2084/transl_table=1misc_feature2160..2161/note="Intron
between 2160 and 2161, approx length 9960 bp" /note="position 2160
corresponds to 21240 in Fig 4 of Anson et al, position 2161
correspnds to 31080 of Fig 4 of Anson et
al"CDS2303..2416/transl_table=1CDS3085..3630/transl_table=1misc_feature33-
98/note="equivalent to position 32318 in Figure 4 of Anson et al
paper" 5ctctctgaca aagatacggt gggtcccact gatgaactgt gctgccacag
taaatgtagc 60cactatgcct atctccattc tgaagatgtg tcacttcctg tttcagactc
aaatcagcca 120cagtggcaga agcccacaga atcagaggtg aaatttaata
atgaccactg cccattctct 180tcacttgtcc caagaggcca ttggaaatag
tccaaagacc cattgaggga gatggacatt 240atttcccaga agtaaataca
gctcagcttg tactttggta caactaatcg accttaccac 300tttcacaact
tgctagcaga ggtt atg cag cgc gtg aac atg atc atg gca 351 Met Gln Arg
Val Asn Met Ile Met Ala 1 5gaa tca cca ggc ctc atc acc atc tgc ctt
tta gga tat cta ctc agt 399Glu Ser Pro Gly Leu Ile Thr Ile Cys Leu
Leu Gly Tyr Leu Leu Ser10 15 20 25gct gaa tgt aca ggtttgtttc
cttttttaaa atacattgag tatgcttgcc 451Ala Glu Cys Thrttttagatat
agaaatatct gatgctgtct tcttcactaa attttgatta catgatttga
511cagcaatatt gaagagtcta acagccagca cgcaggttgg taagtactgg
ttctttgtta 571gctaggtttt cttcttcttc atttttaaaa atctccatgt
gtatacagta ctgtgggaac 631atcacagatt ttggctccta tgccctaaag
agaaattggc tttcagatta tttggattaa 691aaacaaagac tttcttaaga
gatgtaaaat tttcatgatg ttttcttttt tgctaaaact 751aaagaattat
tcttttacat ttca gtt ttt ctt gat cat gaa aac gcc aac 802 Val Phe Leu
Asp His Glu Asn Ala Asn 30 35aaa att ctg aat cgg cca aag agg tat
aat tca ggt aaa ttg gaa gag 850Lys Ile Leu Asn Arg Pro Lys Arg Tyr
Asn Ser Gly Lys Leu Glu Glu 40 45 50ttt gtt caa ggg aac ctt gag aga
gaa tgt atg gaa gaa aag tgt agt 898Phe Val Gln Gly Asn Leu Glu Arg
Glu Cys Met Glu Glu Lys Cys Ser55 60 65 70ttt gaa gaa gca cga gaa
gtt ttt gaa aac act gaa aga aca 940Phe Glu Glu Ala Arg Glu Val Phe
Glu Asn Thr Glu Arg Thr 75 80gtgagtattt ccacataata cccttcagat
gcagagcata gaatagaaaa tctttaaaaa 1000gacacttctc tttaaaattt
taaagcatcc atatatattt atgtatgtta aatgttataa 1060aagataggaa
atcaatacca aaacacttta gatattaccg ttaatttgtc ttcttttatt 1120ctttatag
act gaa ttt tgg aag cag tat gtt ggtaagcaat tcattttatc 1172 Thr Glu
Phe Trp Lys Gln Tyr Val 85 90ctctagctaa tatatgaaac atatgagaca
ggggaggacc gggcattcta agcagtttac 1232gtgccaattc aatttcttaa
cctatctcaa a gat gga gat cag tgt gag tcc 1284 Asp Gly Asp Gln Cys
Glu Ser 95aat cca tgt tta aat ggc ggc agt tgc aag gat gac att aat
tcc tat 1332Asn Pro Cys Leu Asn Gly Gly Ser Cys Lys Asp Asp Ile Asn
Ser Tyr100 105 110 115gaa tgt tgg tgt ccc ttt gga ttt gaa gga aag
aac tgt gaa tta 1377Glu Cys Trp Cys Pro Phe Gly Phe Glu Gly Lys Asn
Cys Glu Leu 120 125 130ggtaagtaac tattttttga atactcatgg ttcaaagttt
ccctctgaaa caagttgaaa 1437ctgaaaattt ctctccccaa cgtatattgg
gggcaacatg aatgccccca atgtatattt 1497gacccataca tgagtcagta
gttccatgta ctttttagaa atgcatgtta aatgatgctg 1557ttactgtcta
ttttgcttct ttta gat gta aca tgt aac att aag aat ggc 1608 Asp Val
Thr Cys Asn Ile Lys Asn Gly 135aga tgc gag cag ttt tgt aaa aat agt
gct gat aac aag gtg gtt tgc 1656Arg Cys Glu Gln Phe Cys Lys Asn Ser
Ala Asp Asn Lys Val Val Cys140 145 150 155tcc tgt act gag gga tat
cga ctt gca gaa aac cag aag tcc tgt gaa 1704Ser Cys Thr Glu Gly Tyr
Arg Leu Ala Glu Asn Gln Lys Ser Cys Glu 160 165 170cca gca
ggtcataatc tgaataagat tttttaaaga aaatctgtat ctgaaacttc 1760Pro
Alaagcattttaa caaacctaca taattttaat tcctacttga cctcaatctc
aatttttgta 1820atacatgttc catttgccaa tgagaaatat caggttacta
atttttcttc tatttttcta 1880gtg cca ttt cca tgt gga aga gtt tct gtt
tca caa act tct aag ctc 1928Val Pro Phe Pro Cys Gly Arg Val Ser Val
Ser Gln Thr Ser Lys Leu 175 180 185acc cgt gct gag gct gtt ttt cct
gat gtg gac tat gta aat tct act 1976Thr Arg Ala Glu Ala Val Phe Pro
Asp Val Asp Tyr Val Asn Ser Thr190 195 200 205gaa gct gaa acc att
ttg gat aac atc act caa agc acc caa tca ttt 2024Glu Ala Glu Thr Ile
Leu Asp Asn Ile Thr Gln Ser Thr Gln Ser Phe 210 215 220aat gac ttc
act cgg gtt gtt ggt gga gaa gat gcc aaa cca ggt caa 2072Asn Asp Phe
Thr Arg Val Val Gly Gly Glu Asp Ala Lys Pro Gly Gln 225 230 235ttc
cct tgg cag gtactttata ctgatggtgt gtcaaaactg gagctcagct 2124Phe Pro
Trp Gln 240ggcaagacac aggccaggtg ggagactgag gctattaaag ctcacatttc
cagaaacatt 2184ccatttctgc cagcacctag aagccaatat tttgcctatt
cctgtaacca gcacacatat 2244ttattttttt ctagatcaaa tgtattatgc
agtaagagtc ttaattttgt tttcacag 2302gtt gtt ttg aat ggt aaa gtt gat
gca ttc tgt gga ggc tct atc gtt 2350Val Val Leu Asn Gly Lys Val Asp
Ala Phe Cys Gly Gly Ser Ile Val 245 250 255aat gaa aaa tgg att gta
act gct gcc cac tgt gtt gaa act ggt gtt 2398Asn Glu Lys Trp Ile Val
Thr Ala Ala His Cys Val Glu Thr Gly Val 260 265 270aaa att aca gtt
gtc gca ggtaaataca cagaaagaat aataatctgc 2446Lys Ile Thr Val Val
Ala 275agcaccacta gctctttaat atgattggta caccatattt tactaaggtc
taataaaatt 2506gttgttgaat aaattgggct aaaggcagaa gggtcataat
ttcagaaccc acgtcgcacc 2566gtcctccaag catccatagt tcttttgata
tacccctatt atcactcatt tcagtgaggt 2626acaattagtt cttgatgtag
ccatttccat accagaaggc cttcccaaaa atcagtgtca 2686tgtcaccgat
ccttttatct ctggtgcttg gcacaacctg tagcaggtcc tcagaaaaca
2746aacatttgaa ttaatggcca aatgagtttg tgctcaaaaa aggggtgagg
atacttgaaa 2806tttggaaaat ctaggataat tcatgactag tggattcatt
atcaccaatg aaaggcttat 2866aacagcatga gtgaacagaa ccatctctat
gatagtcctg aatggctttt tggtctgaaa 2926aatatgcatt ggctctcatt
acatttaacc aaaattatca caatataaga atgagatctt 2986taacattgcc
aattaggtca gtggtcccaa gtagtcactt agaaaatctg tgtatgtgaa
3046atactgtttg tgacttaaaa tgaaatttat ttttaata ggt gaa cat aat att
gag 3102 Gly Glu His Asn Ile Glu 280 285gag aca gaa
cat aca gag caa aag cga aat gtg att cga att att cct 3150Glu Thr Glu
His Thr Glu Gln Lys Arg Asn Val Ile Arg Ile Ile Pro 290 295 300cac
cac aac tac aat gca gct att aat aag tac aac cat gac att gcc 3198His
His Asn Tyr Asn Ala Ala Ile Asn Lys Tyr Asn His Asp Ile Ala 305 310
315ctt ctg gaa ctg gac gaa ccc tta gtg cta aac agc tac gtt aca cct
3246Leu Leu Glu Leu Asp Glu Pro Leu Val Leu Asn Ser Tyr Val Thr Pro
320 325 330att tgc att gct gac aag gaa tac acg aac atc ttc ctc aaa
ttt gga 3294Ile Cys Ile Ala Asp Lys Glu Tyr Thr Asn Ile Phe Leu Lys
Phe Gly 335 340 345tct ggc tat gta agt ggc tgg gga aga gtc ttc cac
aaa ggg aga tca 3342Ser Gly Tyr Val Ser Gly Trp Gly Arg Val Phe His
Lys Gly Arg Ser350 355 360 365gct tta gtt ctt cag tac ctt aga gtt
cca ctt gtt gac cga gcc aca 3390Ala Leu Val Leu Gln Tyr Leu Arg Val
Pro Leu Val Asp Arg Ala Thr 370 375 380tgt ctt cga tct aca aag ttc
acc atc tat aac aac atg ttc tgt gct 3438Cys Leu Arg Ser Thr Lys Phe
Thr Ile Tyr Asn Asn Met Phe Cys Ala 385 390 395ggc ttc cat gaa gga
ggt aga gat tca tgt caa gga gat agt ggg gga 3486Gly Phe His Glu Gly
Gly Arg Asp Ser Cys Gln Gly Asp Ser Gly Gly 400 405 410ccc cat gtt
act gaa gtg gaa ggg acc agt ttc tta act gga att att 3534Pro His Val
Thr Glu Val Glu Gly Thr Ser Phe Leu Thr Gly Ile Ile 415 420 425agc
tgg ggt gaa gag tgt gca atg aaa ggc aaa tat gga ata tat acc 3582Ser
Trp Gly Glu Glu Cys Ala Met Lys Gly Lys Tyr Gly Ile Tyr Thr430 435
440 445aag gta tcc cgg tat gtc aac tgg att aag gaa aaa aca aag ctc
act 3630Lys Val Ser Arg Tyr Val Asn Trp Ile Lys Glu Lys Thr Lys Leu
Thr 450 455 460taatgaaaga tggatttcca aggttaattc attggaattg
aaaattaaca gggcctctca 3690ctaactaatc actttcccat cttttgttag
atttgaatat atacattcta tgatcattgc 3750tttttctctt tacaggggag
aatttcatat tttacctgag caaattgatt agaaaatgga 3810accactagag
gaatataatg tgttaggaaa ttacagtcat ttctaagggc ccagcccttg
3870acaaaattgt gaagttaaat tctccactct gtccatcaga tactatggtt
ctccactatg 3930gcaactaact cactcaattt tccctcctta gcagcattcc
atcttcccga tcttctttgc 3990ttctccaacc aaaacatcaa tgtttattag
ttctgtatac agtacaggat ctttggtcta 4050ctctatcaca aggccagtac
cacactcatg aagaaagaac acaggagtag ctgagaggct 4110aaaactcatc
aaaaacacta ctccttttcc tctaccctat tcctcaatct tttacctttt
4170ccaaatccca atccccaaat cagtttttct ctttcttact ccctctctcc
cttttaccct 4230ccatggtcgt taaaggagag atggggagca tcattctgtt
atacttctgt acacagttat 4290acatgtctat caaacccaga cttgcttcca
tagtggggac ttgcttttca gaacataggg 4350atgaagtaag gtgcctgaaa
agtttggggg aaaagtttct ttcagagagt taagttattt 4410tatatatata
atatatatat aaaatatata atatacaata taaatatata gtgtgtgtgt
4470gtatgcgtgt gtgtagacac acacgcatac acacatataa tggaagcaat
aagccattct 4530aagagcttgt atggttatgg aggtctgact aggcatgatt
tgacgaaggc aagattggca 4590tatcattgta actaaaaaag ctgacattga
cccagacata ttgtactctt tctaaaaata 4650ataataataa tgctaacaga
aagaagagaa ccgttcgttt gcaatctaca gctagtagag 4710actttgagga
agaattcaac agtgtgtctt cagcagtgtt cagagccaag caagaagttg
4770aagttgccta gaccagagga cataagtatc atgtctcctt taactagcat
accccgaagt 4830ggagaagggt gcagcaggct caaaggcata agtcattcca
atcagccaac taagttgtcc 4890ttttctggtt tcgtgttcac catggaacat
tttgattata gttaatcctt ctatcttgaa 4950tcttctagag agttgctgac
caactgacgt atgtttccct ttgtgaatta ataaactggt 5010gttctggttc
ataccttggc tttttgtgga ttccattgat gtgaatcagt caccctgtat
5070ttgatgatgc atgggactac tgacaaaatc actctgactc tgaccctgcc
aagctgctgc 5130cttctcctgc cccaacctca cccccagcca ggcctcactc
tttgctagtt cctttagtct 5190tttagtcaat atatttttgt cttcgcatat
aagtataaat aaacatattt ttaaatttct 5250ggctgggccc agtggctcac
gcctataatc 528068PRTHomo sapiens[CDS]1129..1152 from SEQ ID NO 5
6Thr Glu Phe Trp Lys Gln Tyr Val1 5729PRTHomo sapiens[CDS]325..411
from SEQ ID NO 5 7Met Gln Arg Val Asn Met Ile Met Ala Glu Ser Pro
Gly Leu Ile Thr1 5 10 15Ile Cys Leu Leu Gly Tyr Leu Leu Ser Ala Glu
Cys Thr 20 25838PRTHomo sapiens[CDS]1264..1377 from SEQ ID NO 5
8Asp Gly Asp Gln Cys Glu Ser Asn Pro Cys Leu Asn Gly Gly Ser Cys1 5
10 15Lys Asp Asp Ile Asn Ser Tyr Glu Cys Trp Cys Pro Phe Gly Phe
Glu 20 25 30Gly Lys Asn Cys Glu Leu 35943PRTHomo
sapiens[CDS]1582..1710 from SEQ ID NO 5 9Asp Val Thr Cys Asn Ile
Lys Asn Gly Arg Cys Glu Gln Phe Cys Lys1 5 10 15Asn Ser Ala Asp Asn
Lys Val Val Cys Ser Cys Thr Glu Gly Tyr Arg 20 25 30Leu Ala Glu Asn
Gln Lys Ser Cys Glu Pro Ala 35 401055PRTHomo sapiens[CDS]776..940
from SEQ ID NO 5 10Val Phe Leu Asp His Glu Asn Ala Asn Lys Ile Leu
Asn Arg Pro Lys1 5 10 15Arg Tyr Asn Ser Gly Lys Leu Glu Glu Phe Val
Gln Gly Asn Leu Glu 20 25 30Arg Glu Cys Met Glu Glu Lys Cys Ser Phe
Glu Glu Ala Arg Glu Val 35 40 45Phe Glu Asn Thr Glu Arg Thr 50
551168PRTHomo sapiens[CDS]1881..2084 from SEQ ID NO 5 11Val Pro Phe
Pro Cys Gly Arg Val Ser Val Ser Gln Thr Ser Lys Leu1 5 10 15Thr Arg
Ala Glu Ala Val Phe Pro Asp Val Asp Tyr Val Asn Ser Thr 20 25 30Glu
Ala Glu Thr Ile Leu Asp Asn Ile Thr Gln Ser Thr Gln Ser Phe 35 40
45Asn Asp Phe Thr Arg Val Val Gly Gly Glu Asp Ala Lys Pro Gly Gln
50 55 60Phe Pro Trp Gln651238PRTHomo sapiens[CDS]2303..2416 from
SEQ ID NO 5 12Val Val Leu Asn Gly Lys Val Asp Ala Phe Cys Gly Gly
Ser Ile Val1 5 10 15Asn Glu Lys Trp Ile Val Thr Ala Ala His Cys Val
Glu Thr Gly Val 20 25 30Lys Ile Thr Val Val Ala 3513182PRTHomo
sapiens[CDS]3085..3630 from SEQ ID NO 5 13Gly Glu His Asn Ile Glu
Glu Thr Glu His Thr Glu Gln Lys Arg Asn1 5 10 15Val Ile Arg Ile Ile
Pro His His Asn Tyr Asn Ala Ala Ile Asn Lys 20 25 30Tyr Asn His Asp
Ile Ala Leu Leu Glu Leu Asp Glu Pro Leu Val Leu 35 40 45Asn Ser Tyr
Val Thr Pro Ile Cys Ile Ala Asp Lys Glu Tyr Thr Asn 50 55 60Ile Phe
Leu Lys Phe Gly Ser Gly Tyr Val Ser Gly Trp Gly Arg Val65 70 75
80Phe His Lys Gly Arg Ser Ala Leu Val Leu Gln Tyr Leu Arg Val Pro
85 90 95Leu Val Asp Arg Ala Thr Cys Leu Arg Ser Thr Lys Phe Thr Ile
Tyr 100 105 110Asn Asn Met Phe Cys Ala Gly Phe His Glu Gly Gly Arg
Asp Ser Cys 115 120 125Gln Gly Asp Ser Gly Gly Pro His Val Thr Glu
Val Glu Gly Thr Ser 130 135 140Phe Leu Thr Gly Ile Ile Ser Trp Gly
Glu Glu Cys Ala Met Lys Gly145 150 155 160Lys Tyr Gly Ile Tyr Thr
Lys Val Ser Arg Tyr Val Asn Trp Ile Lys 165 170 175Glu Lys Thr Lys
Leu Thr 180
* * * * *