U.S. patent application number 12/421425 was filed with the patent office on 2009-10-15 for lyophilized dna formulations for enhanced expression of plasmid dna.
This patent application is currently assigned to VIROMED CO., LTD.. Invention is credited to WOONG HAHN, JONG-MOOK KIM, SUJEONG KIM, WONSUN YOO.
Application Number | 20090258932 12/421425 |
Document ID | / |
Family ID | 41162399 |
Filed Date | 2009-10-15 |
United States Patent
Application |
20090258932 |
Kind Code |
A1 |
KIM; JONG-MOOK ; et
al. |
October 15, 2009 |
Lyophilized DNA Formulations for Enhanced Expression of Plasmid
DNA
Abstract
The present invention provides for a method of treating or
preventing ischemic or liver disease in a subject by administering
a composition reconstituted from a lyophilized hepatocyte growth
factor (HGF) DNA formulation, where the DNA formulation comprises
an HGF plasmid DNA, salt and a carbohydrate. The invention further
provides for a method of making such a lyophilized DNA formulation
that preserves or enhances gene expression both in vitro and in
vivo, thus maintaining or stimulating the biological activity of
the expressed protein. The invention also provides for the DNA
formulation, or the lyophilized DNA formulation according to the
methods disclosed.
Inventors: |
KIM; JONG-MOOK; (SEOUL,
KR) ; KIM; SUJEONG; (SEOUL, KR) ; HAHN;
WOONG; (GOYANG-SI, KR) ; YOO; WONSUN; (SEOUL,
KR) |
Correspondence
Address: |
STERNE, KESSLER, GOLDSTEIN & FOX P.L.L.C.
1100 NEW YORK AVENUE, N.W.
WASHINGTON
DC
20005
US
|
Assignee: |
VIROMED CO., LTD.
|
Family ID: |
41162399 |
Appl. No.: |
12/421425 |
Filed: |
April 9, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61043605 |
Apr 9, 2008 |
|
|
|
Current U.S.
Class: |
514/44R |
Current CPC
Class: |
C07K 14/4753 20130101;
A61P 9/10 20180101; A61P 1/16 20180101 |
Class at
Publication: |
514/44.R |
International
Class: |
A61K 31/7088 20060101
A61K031/7088 |
Claims
1. A DNA formulation comprising a plasmid DNA, a salt and a
carbohydrate, wherein said plasmid DNA comprises an HGF gene, or
variant thereof.
2. The DNA formulation of claim 1, wherein said carbohydrate is a
mono-, oligo-, or polysaccharide selected from the group consisting
of sucrose, glucose, lactose, trehalose, arabinose, pentose,
ribose, xylose, galactose, hexose, idose, mannose, talose, heptose,
fructose, gluconic acid, sorbitol, mannitol, methyl
.alpha.-glucopyranoside, maltose, lactone, sorbose, glucaric acid,
erythrose, threose, allose, altrose, gulose, erythrulose, ribulose,
xylulose, psicose, tagatose, glucuronic acid, galacturonic acid,
mannuronic acid, glucosamine, galactosamine, neuraminic acid,
arabinans, fructans, fucans, galactans, galacturonans, glucans,
mannans, xylans, levan, fucoidan, carrageenan, galactocarolose,
pectins, pectic acids, amylose, pullulan, glycogen, amylopectin,
cellulose, dextran, pustulan, chitin, agarose, keratin,
chondroitin, dermatan, hyaluronic acid, alginic acid, xantham gum,
starch, and admixtures thereof.
3. The DNA formulation of claim 1, wherein said carbohydrate is in
an amount selected from the group consisting of between about 0.05%
to about 30%, between about 0.1% to about 15%, between about 0.2%
to about 10%, between about 0.5% and 5%, between about 0.75% and
3%, between about 0.8% and 2%, and between about 0.8% and 1.5%.
4. The DNA formulation of claim 3, wherein said carbohydrate is in
an amount of about 0.1% to about 10%.
5. (canceled)
6. The DNA formulation of claim 2, wherein said carbohydrate is
selected from the group consisting of sucrose, mannitol, and
admixture thereof.
7. The DNA formulation of claim 1, wherein said salt is selected
from the group consisting of NaCl, KCl, and admixture thereof.
8. The DNA formulation of claim 1, wherein said salt is in an
amount selected from the group consisting of between about 0.001%
and about 10%, between about 0.1% and 5%, between about 0.5% and
2%, between about 0.8% and 1.5%, and between about 0.8% and
1.2%.
9. (canceled)
10. The DNA formulation of claim 1, wherein said HGF gene or
variant thereof is selected from the group consisting of flHGF,
dHGF, NK1, NK2, NK4, and admixtures thereof.
11. The DNA formulation of claim 1, wherein said plasmid DNA
comprises a hybrid HGF gene.
12. The DNA formulation of claim 11, wherein said hybrid HGF gene
is selected from the group consisting of HGF-X2, HGF-X3, HGF-X6,
HGF-X7, and HGF-X8.
13. The DNA formulation of claim 12, wherein said plasmid DNA is
selected from the group consisting of: pCK-HGF-X2, pCK-HGF-X3,
pCK-HGF-X6, pCK-HGF-X7, pCK-HGF-X8, pCP-HGF-X2, pCP-HGF-X3,
pCP-HGF-X6, pCP-HGF-X7 and pCP-HGF-X8.
14. The DNA formulation of claim 1, wherein said plasmid DNA is at
a concentration of about 1 ng/mL to about 30 mg/mL.
15. (canceled)
16. (canceled)
17. The DNA formulation of claim 1, wherein said DNA formulation is
lyophilized.
18. The lyophilized DNA formulation of claim 17, wherein the
lyophilization of said DNA formulation comprises (a) loading a
container with said DNA formulation into a lyophilizer; (b) cooling
said DNA formulation to a subzero temperature; and (c) drying said
DNA formulation.
19-21. (canceled)
22. A method of treating or preventing ischemic or liver disease in
a subject, comprising administering a composition reconstituted
from a lyophilized DNA formulation, wherein said lyophilized DNA
formulation comprises a plasmid DNA, a salt and a carbohydrate; and
wherein said plasmid DNA comprises an HGF gene, or variant
thereof.
23-37. (canceled)
38. The method of claim 22, wherein said lyophilized DNA is
reconstituted in a pharmaceutically acceptable solution.
39. The method of claim 38, wherein said pharmaceutically
acceptable solution is selected from the group consisting of water,
PBS, TE, Tris buffer, normal saline, and admixtures thereof.
40. The method of claim 22, wherein said reconstituted composition
is administered by direct injection.
41. A method of making a lyophilized DNA formulation comprising:
(a) preparing a DNA formulation comprising a plasmid DNA, a salt
and a carbohydrate, wherein said plasmid DNA comprises an HGF gene,
or variant thereof; and (b) lyophilizing said DNA formulation,
thereby making said lyophilized DNA formulation.
42. The method of claim 41, wherein the lyophilization of said DNA
formulation further comprises (a) loading a container with said DNA
formulation into a lyophilizer; (b) cooling said DNA formulation to
a subzero temperature; and (c) drying said DNA formulation.
43. (canceled)
44. (canceled)
Description
[0001] This application claims priority to U.S. Provisional Appl.
No. 61/043,605, filed on Apr. 9, 2008, the entire contents of which
are hereby incorporated by reference in their entirety.
REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA
EFS-WEB
[0002] The content of the electronically submitted sequence listing
(Name: Sequence_Listing_Ascii.txt, Size: 68,654 bytes; and Date of
Creation: Apr. 8, 2009) filed herewith the application is
incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
[0003] Lyophilization is often a preferred formulation for
therapeutic materials because the long-term stability of many
materials increases in the lyophilized state. However, for plasmid
DNA, lyophilized formulations are not the formulations of choice.
In most clinical trials using naked (non-complexed plasmid) DNA as
a delivery vector, the preferred formulation has been a liquid
formulation.
[0004] While lyophilized plasmid DNA may be a preferred form of
storage, lyophilized formulations for plasmid DNA have been
considered to cause a reduction in gene expression efficiency.
Lyophilization causes the removal of the hydration sphere around a
molecule. For DNA, it appears that there are approximately 20 water
molecules per nucleotide pair bound most tightly to DNA that do not
form an ice-like structure upon low-temperature cooling. Upon DNA
dehydration over hygroscopic salts at 0% relative humidity, only
five or six water molecules remain. Thus, lyophilization may
increase the stability of DNA under long-term storage, but may also
cause some damage upon the initial lyophilization process,
potentially through changes in the DNA secondary structure or the
concentration of reactive elements such as contaminating metals.
Therefore, a potential mechanism for loss of gene expression
efficiency of lyophilized plasmid DNA may be through a gross
structural change to the plasmid.
[0005] In Poxon et al, Pharmaceutical Development and Technology
5:115-122 (2000), the authors demonstrated that lyophilization of a
plasmid DNA (pRL-CMV) resulted in a statistically significant loss
of transfection efficiency. A biofunctionality assay, measuring
transfection activity, demonstrated a loss of more than 75% of
plasmid DNA activity after lyophilization as compared to control
plasmid that remained in solution. While Poxon et al used
carbohydrates to ameliorate the in vitro decreased transfection
activity of a non-therapeutic plasmid, pRL-CMV expressing Renilla
luciferase, stored in EDTA buffer, Poxon et al did not address the
use of lyophilized naked DNA formulations in vivo for disease
treatment or prevention.
[0006] Therefore, there is a need in the art for a stable
lyophilized formulation that will not affect gene expression
efficiency. The present invention provides for a lyophilized
formulation for plasmid DNA that not only preserves the biological
activity of the expressed gene but, in certain instances, is able
to enhance biological activity.
BRIEF SUMMARY OF THE INVENTION
[0007] The present invention is directed to a lyophilized DNA
formulation. In one aspect of the invention, a DNA formulation,
prior to lyophilization, comprises a plasmid DNA, salt and a
carbohydrate; and where the plasmid DNA comprises an HGF gene, or
variant thereof. In another aspect of the invention, the DNA
formulation is lyophilized. In another aspect of the invention, the
lyophilized DNA formulation is reconstituted.
[0008] In one embodiment, the carbohydrate of the DNA formulation
of the present invention is a mono-, oligo-, or polysaccharide such
as sucrose, glucose, lactose, trehalose, arabinose, pentose,
ribose, xylose, galactose, hexose, idose, mannose, talose, heptose,
fructose, gluconic acid, sorbitol, mannitol, methyl
.alpha.-glucopyranoside, maltose, isoascorbic acid, ascorbic acid,
lactone, sorbose, glucaric acid, erythrose, threose, allose,
altrose, gulose, erythrulose, ribulose, xylulose, psicose,
tagatose, glucuronic acid, galacturonic acid, mannuronic acid,
glucosamine, galactosamine, neuraminic acid, arabinans, fructans,
fucans, galactans, galacturonans, glucans, mannans, xylans, levan,
fucoidan, carrageenan, galactocarolose, pectins, pectic acids,
amylose, pullulan, glycogen, amylopectin, cellulose, dextran,
cyclodextrin, pustulan, chitin, agarose, keratin, chondroitin,
dermatan, hyaluronic acid, alginic acid, xantham gum, or
starch.
[0009] In certain embodiments of the invention, the carbohydrate is
sucrose or mannitol.
[0010] In another embodiment, the carbohydrate of the DNA
formulation of the present invention is in an amount selected from
the group consisting of between about 0.05% to about 30%, between
about 0.1% to about 15%, between about 0.2% to about 10%, between
about 0.5% and 5%, between about 0.75% and 3%, between about 0.8%
and 2%, and between about 0.8% and 1.5%. In particular embodiments,
the carbohydrate is sucrose or mannitol. In certain other
embodiments, the carbohydrate of the DNA formulation is in an
amount of about 1.1%.
[0011] In another embodiment, the salt of the DNA formulation is
selected from the group consisting of NaCl or KCl. In further
embodiments, the salt of the DNA formulation is in an amount
selected from the group consisting of between about 0.01% and 10%,
between about 0.1% and 5%, between about 0.1% and 4%, between about
0.5% and 2%, between about 0.8% and 1.5%, between about 0.8% and
1.2% w/v. In certain embodiments, the salt of the DNA formulation
is in an amount of about 0.9% w/v.
[0012] In another embodiment, the plasmid DNA of the invention
comprises an HGF gene, or variant thereof. In certain embodiments,
the HGF gene is a mammalian HGF gene or variant thereof. In further
embodiments, the HGF gene is a human HGF gene or variant thereof.
In certain aspects of the invention, the HGF gene is a hybrid HGF
gene, e.g., a hybrid HGF gene comprising HGF cDNA and an inherent
or foreign intron or fragment thereof, e.g., an inherent intron 4
or fragment thereof of the human HGF gene. In particular
embodiments, the hybrid HGF gene comprises HGF-X2 (SEQ ID NO: 13),
HGF-X3 (SEQ ID NO: 14), HGF-X6 (SEQ ID NO: 8), HGF-X7 (SEQ ID NO:
9) or HGF-X8 (SEQ ID NO: 10). In further embodiments, the plasmid
DNA comprising a hybrid HGF gene is selected from the group
consisting of: pCK-HGF-X2, pCK-HGF-X3, pCK-HGF-X6, pCK-HGF-X7,
pCK-HGF-X8, pCP-HGF-X2, pCP-HGF-X3, pCP-HGF-X6, pCP-HGF-X7 and
pCP-HGF-X8, where the HGF-X2, HGF-X3, HGF-X6, HGF-X7 and HGF-X8
correspond to SEQ ID NOs: 13-14 and 8-10, respectively.
[0013] The lyophilized DNA formulations maintain or enhance the
expression of the plasmid DNA. In certain aspects, the lyophilized
DNA formulation provides enhanced biological activity of the
expressed protein. In certain other aspects of the invention, the
enhanced expression of the plasmid DNA or the enhanced biological
activity of the expressed protein is due to the presence of the
carbohydrate in the formulation. In certain embodiments, this
carbohydrate is sucrose or mannitol.
[0014] The invention also provides for a reconstituted lyophilized
plasmid DNA formulation. In certain embodiments, the lyophilized
DNA is reconstituted in a pharmaceutically acceptable solution. In
further embodiments, the pharmaceutically acceptable solution is
selected from the group consisting of water, PBS, TE, Tris buffer
and normal saline.
[0015] In another embodiment, the plasmid DNA of the reconstituted
lyophilized formulation is at a final concentration of about 1
ng/mL, about 5 ng/mL, about 10 ng/mL, about 50 ng/mL, about 100
ng/mL, about 250 ng/mL, about 500 ng/mL, about 1 .mu.g/mL, about 5
.mu.g/mL, about 10 .mu.g/mL, about 50 .mu.g/mL, about 100 .mu.g/mL,
about 200 .mu.g/mL, about 300 .mu.g/mL, about 400 .mu.g/mL, about
500 .mu.g/mL, about 600 .mu.g/mL, about 700 .mu.g/mL, about 800
.mu.g/mL, about 900 .mu.g/mL, about 1 mg/mL, about 2 mg/mL, about
2.5 mg/mL, about 3 mg/mL, about 3.5 mg/mL, about 4 mg/mL, about 4.5
mg/mL, about 5 mg/mL, about 5.5 mg/mL, about 6 mg/mL, about 7
mg/mL, about 8 mg/mL, about 9 mg/mL, about 10 mg/mL, about 20
mg/mL, or about 30 mg/mL. In another embodiment, the final
concentration of the plasmid DNA of the reconstituted lyophilized
formulation is from about 1 ng/mL to about 30 mg/mL. In certain
aspects, the final concentration of the plasmid DNA of the
reconstituted lyophilized formulation is from about 100 .mu.g/mL to
about 2.5 mg/mL. In further aspects, the final concentration of the
plasmid DNA of the reconstituted lyophilized formulation is from
about 500 .mu.g/mL to about 1 mg/mL.
[0016] The present invention is also directed to a method of
treating or preventing ischemic or liver disease in a subject,
comprising administering a composition reconstituted from a
lyophilized hepatocyte growth factor (HGF) DNA formulation, where
the DNA formulation comprises a plasmid DNA, salt and a
carbohydrate; and where the plasmid DNA comprises an HGF gene, or
variant thereof. In certain aspects, the composition reconstituted
from a lyophilized HGF DNA formulation is administered by direct
injection.
[0017] The present invention is further directed to a method of
making a lyophilized HGF DNA formulation comprising: (a) preparing
a DNA formulation comprising a plasmid DNA, a salt and a
carbohydrate, where the plasmid DNA comprises an HGF gene, or
variant thereof; and (b) lyophilizing the DNA formulation.
[0018] The steps for lyophilization may include subjecting a DNA
formulation of the invention to the process of being frozen at
subzero temperatures (e.g, -10.degree. C. to -500.degree. C.), and
then subjected to one or more drying cycles which comprises
gradually heating the DNA formulation to a temperature of about
20.degree. C. to less than or equal to about 30.degree. C., wherein
the lyophilization occurs over a period of about 50 to about 100
hours. In a further aspect of the invention, the method for
lyophilization comprises: (a) forming an aqueous DNA formulation
comprising a plasmid DNA, a salt and a carbohydrate, where the
plasmid DNA comprises an HGF gene, or variant thereof; (b) cooling
the DNA formulation solution to a temperature of about -10.degree.
C. to about -50.degree. C., until frozen; (c) drying the DNA
formulation by heating to a temperature of about 20.degree. C. to
about 30.degree. C.; and (d) recovering a lyophilized DNA
formulation composition having a water content of from about 0.1
weight percent to about 5 weight percent based on the total weight
of the recovered DNA formulation.
[0019] In certain embodiments, the DNA formulation is lyophilized
under conditions comprising (a) about 30 hours to about 50 hours at
a temperature greater than or equal to about -50.degree. C. and
less than about 0.degree. C., and (b) about 20 hours to about 50
hours at a temperature greater than or equal to about 0.degree. C.
to less than or equal to about 30.degree. C., progressively,
wherein the lowest (a) temperature is about -50.degree. C. to about
-30.degree. C. and the highest (b) temperature is between about
20.degree. C. to about 30.degree. C. In one aspect, the DNA
formulation is lyophilized under conditions of -50.degree. C. for 4
hours, -40.degree. C. for 12 hours, -30.degree. C. for 6 hours,
-20.degree. C. for 6 hours, -10.degree. C. for 6 hours, 0.degree.
C. for 6 hours, 10.degree. C. for 6 hours and 30.degree. C. for 24
hours, progressively. In another aspect, the DNA formulation is
lyophilized under conditions of 5.degree. C. for 1 minute,
-50.degree. C. for 2 hours, -40.degree. C. for 6 hours, -35.degree.
C. for 3 hours, -30.degree. C. for 6 hours, -25.degree. C. for 3
hours, -20.degree. C. for 3 hours, -15.degree. C. for 3 hours,
-10.degree. C. for 6 hours, -5.degree. C. for 3 hours, 0.degree. C.
for 6 hours, and 30.degree. C. for 17 hours, progressively. In
another aspect, the DNA formulation is lyophilized under conditions
of 5.degree. C. for 1 minute, -10.degree. C. for 1 minute,
-20.degree. C. for 1 minute, -30.degree. C. for 1 minute,
-50.degree. C. for 1 minute, -50.degree. C. for 2 hours,
-45.degree. C. for 6 hours, -40.degree. C. for 3 hours, -35.degree.
C. for 6 hours, -30.degree. C. for 3 hours, -25.degree. C. for 6
hours, -20.degree. C. for 3 hours, -15.degree. C. for 6 hours,
-10.degree. C. for 3 hours, -5.degree. C. for 6 hours, 0.degree. C.
for 12 hours, 10.degree. C. for 3 hours, 20.degree. C. for 6 hours,
and 30.degree. C. for 29 hours, progressively.
[0020] The invention is further directed to a lyophilized nucleic
acid formulation or a reconstituted lyophilized nucleic acid
formulation, as set forth above, where the nucleic acid is an RNA
that encodes for HGF, or variant thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 depicts a bar graph comparing in vitro HGF expression
among various formulations. HGF expression levels were measured
using ELISA in culture supernatants isolated from 293T cells
transfected with a lyophilized plasmid DNA pCK-HGF-X7 formulated in
0.9% NaCl at a final DNA concentration of 0.5 mg/mL, with sucrose
at 0.25% (lane 3), 1.1% (lane 4), 5% (lane 5), 10% (lane 6) or 20%
(lane 7) or with mannitol at 1.2% (lane 8), 4.85% (lane 9) or 10%
(lane 10). Control reactions with a negative control (lane 1) and
non-lyophilized DNA (lane 2) were used as comparison.
[0022] FIG. 2 depicts a bar graph comparing in vivo HGF expression
between non-lyophilized and lyophilized pCK-HGF-X7. Mice were
injected with 100 .mu.g of non-lyophilized pCK-HGF-X7 containing
0.9% NaCl (NL-HGF-X7) or pCK-HGF-X7 lyophilized with 1.1% Sucrose
and 0.9% NaCl (L-HGF-X7) into the tibialis cranialis. HGF
expression levels were measured using ELISA in muscle tissue
lysates after sacrificing the mice at day 7. HGF expression levels
are shown for negative control (lane 1), non-lyophilized pCK-HGF-X7
containing 0.9% NaCl (NL-HGF-X7; lane 2), and pCK-HGF-X7
lyophilized with 1.1% sucrose and 0.9% NaCl (L-HGF-X7; lane 3).
[0023] FIG. 3 shows a schematic diagram of the experimental
procedure using the porcine ischemic heart disease model. NL-HGF-X7
corresponds to non-lyophilized pCK-HGF-X7 containing 0.9% NaCl.
L-HGF-X7 corresponds to pCK-HGF-X7 lyophilized with 1.1% sucrose
and 0.9% NaCl.
[0024] FIG. 4 depicts a bar graph showing the effect of
non-lyophilized and lyophilized pCK-HGF-X7 on myocardial perfusion.
The percent improvement of myocardial perfusion as compared to
baseline is shown when the porcine ischemic heart disease model is
utilized. Results are shown for pigs injected with plasmid alone
(pCK; lane 1), non-lyophilized pCK-HGF-X7 containing 0.9% NaCl
(NL-HGF-X7; lane 2), and pCK-HGF-X7 lyophilized with 1.1% sucrose
and 0.9% NaCl (L-HGF-X7; lane 3).
[0025] FIG. 5 depicts a bar graph showing the effect of
non-lyophilized and lyophilized pCK-HGF-X7 on wall thickening. The
percent improvement on wall thickening in the injected ischemic
border area of the left ventricle as compared to baseline is shown
when the porcine ischemic heart disease model is utilized. Results
are shown for pigs injected with plasmid alone (pCK; lane 1),
non-lyophilized pCK-HGF-X7 containing 0.9% NaCl (NL-HGF-X7; lane
2), and pCK-HGF-X7 lyophilized with 1.1% sucrose and 0.9% NaCl
(L-HGF-X7; lane 3).
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0026] The term "DNA" or "nucleic acid" or "nucleic acid fragment"
refers to any one or more nucleic acid segments, e.g., DNA or RNA
fragments, present in a polynucleotide or construct. A nucleic acid
or fragment thereof may be provided in linear (e.g., mRNA) or
circular (e.g., plasmid) form as well as double-stranded or
single-stranded forms. By "isolated" nucleic acid or polynucleotide
is intended a nucleic acid molecule, DNA or RNA, which has been
removed from its native environment. For example, a recombinant
polynucleotide contained in a vector is considered isolated for the
purposes of the present invention. Further examples of an isolated
polynucleotide include recombinant polynucleotides maintained in
heterologous host cells or purified (partially or substantially)
polynucleotides in solution. Isolated RNA molecules include in vivo
or in vitro RNA transcripts of the polynucleotides of the present
invention. Isolated polynucleotides or nucleic acids according to
the present invention further include such molecules produced
synthetically.
[0027] As used herein, a "coding region" is a portion of nucleic
acid which consists of codons translated into amino acids. Although
a "stop codon" (TAG, TGA, or TAA) is not translated into an amino
acid, it may be considered to be part of a coding region, but any
flanking sequences, for example promoters, ribosome binding sites,
transcriptional terminators, and the like, are not part of a coding
region. Two or more nucleic acids or nucleic acid fragments of the
present invention can be present in a single polynucleotide
construct, e.g., on a single plasmid, or in separate polynucleotide
constructs, e.g., on separate (different) plasmids. Furthermore,
any nucleic acid or nucleic acid fragment may encode a single HGF
polypeptide or fragment, derivative, or variant thereof, e.g., or
may encode more than one polypeptide, e.g., a nucleic acid may
encode two or more polypeptides. In addition, a nucleic acid may
include a regulatory element such as a promoter, ribosome binding
site, or a transcription terminator, or may encode heterologous
coding regions fused to the HGF coding region, e.g., specialized
elements or motifs, such as a secretory signal peptide or a
heterologous functional domain.
[0028] In the case of DNA, a polynucleotide comprising a nucleic
acid which encodes a polypeptide normally also comprises a promoter
and/or other transcription or translation control elements operably
associated with the polypeptide-encoding nucleic acid fragment. An
operable association is when a nucleic acid fragment encoding a
gene product, e.g., a polypeptide, is associated with one or more
regulatory sequences in such a way as to place expression of the
gene product under the influence or control of the regulatory
sequence(s).
[0029] A DNA polynucleotide of the present invention may be a
circular or linearized plasmid or vector, or other linear DNA which
may also be non-infectious and nonintegrating (i.e., does not
integrate into the genome of vertebrate cells). A linearized
plasmid is a plasmid that was previously circular but has been
linearized, for example, by digestion with a restriction
endonuclease. As used herein, the terms plasmid and vector can be
used interchangeably.
[0030] The term "lyophilized DNA" refers to any DNA that is
prepared in dry form by rapid freezing and dehydration, in the
frozen state under high vacuum. "Lyophilizing" or "lyophilization"
refers to a process of freezing and drying a solution. Lyophilized
DNA is often made ready for use by addition of sterile distilled
water.
[0031] A "vector" refers to any vehicle for the cloning of and/or
transfer of a nucleic acid into a host cell. A vector may be a
replicon to which another DNA segment may be attached so as to
bring about the replication of the attached segment. A "replicon"
refers to any genetic element (e.g., plasmid, phage, cosmid,
chromosome, virus) that functions as an autonomous unit of DNA
replication in vivo, i.e., capable of replication under its own
control. The term "vector" includes vehicles for introducing the
nucleic acid into a cell in vitro, ex vivo or in vivo. A large
number of vectors known in the art may be used to manipulate
nucleic acids, incorporate response elements and promoters into
genes, etc. Possible vectors include, for example, plasmids such as
pBR322 or pUC plasmid derivatives, or the Bluescript vector. For
example, the insertion of the DNA fragments corresponding to
response elements and promoters into a suitable vector can be
accomplished by ligating the appropriate DNA fragments into a
chosen vector that has complementary cohesive termini.
Alternatively, the ends of the DNA molecules may be enzymatically
modified or any site may be produced by ligating nucleotide
sequences (linkers) into the DNA termini. Such vectors may be
engineered to contain selectable marker genes that provide for the
selection of cells. Such markers allow identification and/or
selection of host cells that express the proteins encoded by the
marker.
[0032] Additional vectors include lipoplexes (cationic liposome-DNA
complex), polyplexes (cationic polymer-DNA complex), and
protein-DNA complexes. In addition to a nucleic acid, a vector may
also comprise one or more regulatory regions, and/or selectable
markers useful in selecting, measuring, and monitoring nucleic acid
transfer results (transfer to which tissues, duration of
expression, etc.).
[0033] The term "plasmid" refers to an extra-chromosomal element
often carrying a gene that is not part of the central metabolism of
the cell, and usually in the form of circular double-stranded DNA
molecules. Such elements may be autonomously replicating sequences,
genome integrating sequences, phage or nucleotide sequences,
linear, circular, or supercoiled, of a single- or double-stranded
DNA or RNA, derived from any source, in which a number of
nucleotide sequences have been joined or recombined into a unique
construction which is capable of introducing a promoter fragment
and DNA sequence for a selected gene product along with appropriate
3' untranslated sequence into a cell. As used herein, the term
"plasmid" refers to a construct made up of genetic material (i.e.,
nucleic acids). Typically a plasmid contains an origin of
replication which is functional in bacterial host cells, e.g.,
Escherichia coli, and selectable markers for detecting bacterial
host cells comprising the plasmid.
[0034] Plasmids of the present invention may include genetic
elements as described herein arranged such that an inserted coding
sequence can be transcribed and translated in eukaryotic cells. In
certain embodiments described herein, a plasmid is a closed
circular DNA molecule.
[0035] The term "expression" refers to the biological production of
a product encoded by a coding sequence. In most cases a DNA
sequence, including the coding sequence, is transcribed to form a
messenger-RNA (mRNA). The messenger-RNA is then translated to form
a polypeptide product which has a relevant biological activity.
Also, the process of expression may involve further processing
steps to the RNA product of transcription, such as splicing to
remove introns, and/or post-translational processing of a
polypeptide product.
[0036] The term "expression vector" refers to a vector, plasmid or
vehicle designed to enable the expression of an inserted nucleic
acid sequence following transformation into the host. The cloned
gene, i.e., the inserted nucleic acid sequence, e.g., a HGF gene or
variant thereof, is usually placed under the control of control
elements such as a promoter, a minimal promoter, an enhancer, or
the like. Initiation control regions or promoters, which are useful
to drive expression of a nucleic acid in the desired host cell are
numerous and familiar to those skilled in the art. Virtually any
promoter capable of driving expression of these genes can be used
in an expression vector, including but not limited to, viral
promoters, bacterial promoters, animal promoters, mammalian
promoters, synthetic promoters, constitutive promoters, tissue
specific promoters, pathogenesis or disease related promoters,
developmental specific promoters, inducible promoters, light
regulated promoters; including, but are not limited to, the SV40
early (SV40) promoter region, the promoter contained in the 3' long
terminal repeat (LTR) of Rous sarcoma virus (RSV), the E1A or major
late promoter (MLP) of adenoviruses (Ad), the human cytomegalovirus
(HCMV) immediate early promoter, the herpes simplex virus (HSV)
thymidine kinase (TK) promoter, the baculovirus IE1 promoter, the
elongation factor 1 alpha (EF1) promoter, the
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, the
phosphoglycerate kinase (PGK) promoter, the ubiquitin C (Ube)
promoter, the albumin promoter, the regulatory sequences of the
mouse metallothionein-L promoter and transcriptional control
regions, the ubiquitous promoters (HPRT, vimentin, .beta.-actin,
tubulin and the like), the promoters of the intermediate filaments
(desmin, neurofilaments, keratin, GFAP, and the like), the
promoters of therapeutic genes (of the MDR, CFTR or factor VIII
type, and the like), pathogenesis or disease related-promoters, and
promoters that exhibit tissue specificity and have been utilized in
transgenic animals, such as the elastase I gene control region
which is active in pancreatic acinar cells; insulin gene control
region active in pancreatic beta cells, immunoglobulin gene control
region active in lymphoid cells, mouse mammary tumor virus control
region active in testicular, breast, lymphoid and mast cells;
albumin gene, Apo AI and Apo AII control regions active in liver,
alpha-fetoprotein gene control region active in liver, alpha
1-antitrypsin gene control region active in the liver, beta-globin
gene control region active in myeloid cells, myelin basic protein
gene control region active in oligodendrocyte cells in the brain,
myosin light chain-2 gene control region active in skeletal muscle,
and gonadotropic releasing hormone gene control region active in
the hypothalamus, pyruvate kinase promoter, villin promoter,
promoter of the fatty acid binding intestinal protein, promoter of
the smooth muscle cell .beta.-actin, and the like. In addition,
these expression sequences may be modified by addition of enhancer
or regulatory sequences and the like. Non-limiting examples of
expression vectors of the invention include pCK (Lee et al.,
Biochem. Biophys. Res. Commun. 272:230 (2000); WO 2000/040737) and
pCP (pcDNA3.1, Invitrogen, USA).
[0037] A "construct" as used herein generally denotes a composition
that does not occur in nature. A construct can be produced by
synthetic technologies, e.g., recombinant DNA preparation and
expression or chemical synthetic techniques for nucleic or amino
acids. A construct can also be produced by the addition or
affiliation of one material with another such that the result is
not found in nature in that form.
[0038] A "gene" refers to a polynucleotide comprising nucleotides
that encode a functional molecule, including functional molecules
produced by transcription only (e.g., a bioactive RNA species) or
by transcription and translation (e.g., a polypeptide). The term
"gene" encompasses cDNA and genomic DNA nucleic acids. "Gene" also
refers to a nucleic acid fragment that expresses a specific RNA,
protein or polypeptide, including regulatory sequences preceding
(5' non-coding sequences) and following (3' non-coding sequences)
the coding sequence. "Native gene" refers to a gene as found in
nature with its own regulatory sequences. "Chimeric gene" refers to
any gene that is not a native gene, comprising regulatory and/or
coding sequences that are not found together in nature.
Accordingly, a chimeric gene may comprise regulatory sequences and
coding sequences that are derived from different sources, or
regulatory sequences and coding sequences derived from the same
source, but arranged in a manner different than that found in
nature. A chimeric gene may comprise coding sequences derived from
different sources and/or regulatory sequences derived from
different sources. "Endogenous gene" refers to a native gene in its
natural location in the genome of an organism. A "foreign" gene or
"heterologous" gene refers to a gene not normally found in the host
organism, but that is introduced into the host organism by gene
transfer. Foreign genes can comprise native genes inserted into a
non-native organism, or chimeric genes. A "transgene" is a gene
that has been introduced into the cell by a gene transfer
procedure.
[0039] "Heterologous DNA" refers to DNA not naturally located in
the cell, or in a chromosomal site of the cell. The heterologous
DNA may include a gene foreign to the cell.
[0040] The phrases "isolated" or "biologically pure" refer to
material that is substantially or essentially free from components
which normally accompany the material as it is found in its native
state. Thus, isolated peptides in accordance with the invention
preferably do not contain materials normally associated with the
peptides in their in situ environment.
Lyophilized DNA formulations
[0041] The DNA formulation of the invention, prior to
lyophilization, is formulated with certain excipients, including a
carbohydrate and a salt.
[0042] As described herein, the stability of a lyophilized
formulation of DNA to be utilized as a diagnostic or therapeutic
agent can be increased by formulating the DNA prior to
lyophilization with an aqueous solution comprising a stabilizing
amount of carbohydrate.
[0043] A carbohydrate of the DNA formulation of the invention is a
mono-, oligo-, or polysaccharide, such as sucrose, glucose,
lactose, trehalose, arabinose, pentose, ribose, xylose, galactose,
hexose, idose, mannose, talose, heptose, fructose, gluconic acid,
sorbitol, mannitol, methyl .alpha.-glucopyranoside, maltose,
isoascorbic acid, ascorbic acid, lactone, sorbose, glucaric acid,
erythrose, threose, allose, altrose, gulose, erythrulose, ribulose,
xylulose, psicose, tagatose, glucuronic acid, galacturonic acid,
mannuronic acid, glucosamine, galactosamine, neuraminic acid,
arabinans, fructans, fucans, galactans, galacturonans, glucans,
mannans, xylans, levan, fucoidan, carrageenan, galactocarolose,
pectins, pectic acids, amylose, pullulan, glycogen, amylopectin,
cellulose, dextran, cyclodextrin, pustulan, chitin, agarose,
keratin, chondroitin, dermatan, hyaluronic acid, alginic acid,
xantham gum, or starch.
[0044] In one aspect, the carbohydrate is mannitol or sucrose.
[0045] The carbohydrate solution prior to lyophilization can
correspond to carbohydrate in water alone, or a buffer can be
included. Examples of such buffers include PBS, HEPES, TRIS or
TRIS/EDTA. Typically the carbohydrate solution is combined with the
DNA to a final concentration of about 0.05% to about 30% sucrose,
typically 0.1% to about 15% sucrose, such as 0.2% to about 5%, 10%
or 15% sucrose, preferably between about 0.5% to 10% sucrose, 1% to
5% sucrose, 1% to 3% sucrose, and most preferably about 1.1%
sucrose.
[0046] A salt of the DNA formulation of the invention is NaCl or
KCl. In certain aspects, the salt is NaCl. In further aspects, the
salt of the DNA formulation is in an amount selected from the group
consisting of between about 0.001% to about 10%, between about 0.1%
and 5%, between about 0.1% and 4%, between about 0.5% and 2%,
between about 0.8% and 1.5%, between about 0.8% and 1.2% w/v. In
certain embodiments, the salt of the DNA formulation is in an
amount of about 0.9% w/v.
[0047] In the DNA formulation of the invention, the final
concentration of DNA is from about 1 ng/mL to about 30 mg/mL of
plasmid. For example, a formulation of the present invention may
have a final concentration of about 1 ng/mL, about 5 ng/mL, about
10 ng/mL, about 50 ng/mL, about 100 ng/mL, about 200 ng/mL, about
500 ng/mL, about 1 .mu.g/mL, about 5 .mu.g/mL, about 10 .mu.g/mL,
about 50 .mu.g/mL, about 100 .mu.g/mL, about 200 .mu.g/mL, about
400 .mu.g/mL, about 500 .mu.g/mL, about 600 .mu.g/mL, about 800
.mu.g/mL, about 1 mg/mL, about 2 mg/mL, about 2.5 mg/mL, about 3
mg/mL, about 3.5 mg/mL, about 4 mg/mL, about 4.5 mg/mL, about 5
mg/mL, about 5.5 mg/mL, about 6 mg/mL, about 7 mg/mL, about 8
mg/mL, about 9 mg/mL, about 10 mg/mL, about 20 mg/mL, or about 30
mg mg/mL of a plasmid. In certain embodiments of the invention, the
final concentration of the DNA is from about 100 .mu.g/mL to about
2.5 mg/mL. In particular embodiments of the invention, the final
concentration of the DNA is from about 0.5 mg/mL to 1 mg/mL.
[0048] The DNA formulation of the invention is lyophilized under
standard conditions known in the art. A method for lyophilization
of the DNA formulation of the invention may comprise (a) loading a
container, e.g., a vial, with a DNA formulation, e.g., a DNA
formulation comprising a plasmid DNA, a salt and a carbohydrate,
where the plasmid DNA comprises an HGF gene, or variant thereof,
into a lyophilizer, wherein the lyophilizer has a starting
temperature of about 5.degree. C. to about -50.degree. C.; (b)
cooling the DNA formulation to subzero temperatures (e.g.,
-10.degree. C. to -50.degree. C.); and (c) substantially drying the
DNA formulation. The conditions for lyophilization, e.g.,
temperature and duration, of the DNA formulation of the invention
can be adjusted by a person of ordinary skill in the art taking
into consideration factors that effect lyophilization parameters,
e.g., the type of lyophilization machine used, the amount of DNA
used, and the size of the container used.
[0049] The container holding the lyophilized DNA formulation may
then be sealed and stored for an extended period of time at various
temperatures (e.g., room temperature to about -180.degree. C.,
preferably about 2-8.degree. C. to about -80.degree. C., more
preferably about -20.degree. C. to about -80.degree. C., and most
preferably about -20.degree. C.). In certain aspects, the
lyophilized DNA formulations are preferably stable within a range
of from about 2-8.degree. C. to about -80.degree. C. for a period
of at least 6 months without losing significant activity. Stable
storage plasmid DNA formulation can also correspond to storage of
plasmid DNA in a stable form for long periods of time before use as
such for research or plasmid-based therapy. Storage time may be as
long as several months, 1 year, 5 years, 10 years, 15 years, or up
to 20 years. Preferably the preparation is stable for a period of
at least about 3 years.
HGF Plasmid DNA
[0050] The present invention provides for a lyophilized DNA
formulation, where the DNA formulation, prior to lyophilization,
comprises a plasmid DNA, and the plasmid DNA comprises an HGF gene,
or variant thereof.
[0051] Hepatocyte growth factor (HGF) is a heparin binding
glycoprotein also known as scatter factor or hepatopoietin-A. An
endogenous gene encoding human HGF is located at chromosome 7q21.1
and comprises 18 exons and 17 introns, having the nucleotide
sequence of SEQ ID NO: 1 (Seki T., et al., Gene 102:213-219
(1991)). A transcript of about 6 kb is transcribed from the HGF
gene, and then, a polypeptide HGF precursor consisting of 728 amino
acids (SEQ ID NO: 2) is synthesized therefrom. Simultaneously, a
polypeptide of dHGF precursor consisting of 723 amino acids is also
synthesized by an alternative splicing of the HGF gene. The
biologically inactive precursors may be converted into active forms
of disulfide-linked heterodimer by protease in serum. In the
heterodimers, the alpha chain having a high molecular weight forms
four kringle domains and an N-terminal hairpin loop like a
preactivated peptide region of plasminogen. The kringle domains of
a triple disulfide-bonded loop structure consisting of about 80
amino acids may play an important role in protein-protein
interaction. The low molecular weight beta chain forms an inactive
serine protease-like domain. dHGF consisting 723 amino acids is a
polypeptide with deletion of five amino acids in the 1st kringle
domain of the alpha chain, i.e., F, L, P, S and S.
[0052] HGF secreted from mesoderm-derived cells has various
biological functions, e.g., 1) inducing epithelial cells into a
tubular structure; 2) stimulating vascularization from endothelial
cells in vitro and in vivo; 3) regeneration of liver and kidney,
owing to its anti-apoptosis activity; 4) organogenesis of kidney,
ovary and testis; 5) controlling osteogenesis; 6) stimulating the
growth and differentiation of erythroid hematopoietic precursor
cells; and 7) axon sprouting of neurons (Stella, M. C. and
Comoglio, P. M., The International Journal of Biochemistry &
Cell Biology 31:1357-1362 (1999)). Based on these various
functions, HGF or a gene encoding HGF or a variant thereof, may be
developed as a therapeutic agent for treating ischemic or liver
diseases. Actually, in vivo, the HGF may exist as either HGF or
dHGF, and therefore, the coexpression of HGF and dHGF is important
for maximizing the therapeutic effect. A hybrid HGF gene which can
simultaneously express HGF and dHGF with a high efficiency for gene
therapy is an HGF variant that would be advantageous to utilize in
the plasmid DNA formulation of the present invention.
[0053] The hybrid HGF gene has been previously described in Intl.
Appl. No. WO 03/078568 and U.S. Publ. No. 2005/0079581 A1, the
contents of each which are herein incorporated by reference. The
hybrid HGF gene is prepared by inserting an inherent or foreign
intron between exons 4 and 5 in HGF cDNA. The hybrid HGF gene has a
higher expression efficiency than HGF cDNA and simultaneously
expresses two heterotypes of HGF and dHGF (deleted variant
HGF).
[0054] The term "isoform of HGF" refers to any HGF polypeptide
having an amino acid sequence that is at least 80% identical (e.g.,
at least 90% or 95% identical) to a HGF amino acid sequence that is
naturally produced in an animal, including all allelic variants. In
one embodiment, the term refers to isoforms that are known to have
cell proliferation activity. Isoforms of HGF include, without
limitation, flHGF, dHGF, NK1, NK2, and NK4, e.g., corresponding to
SEQ ID NOs: 2-6, and variants thereof (e.g., NK2 variants, SEQ ID
NOs: 11-12).
[0055] The term "flHGF" refers to the full length HGF protein of an
animal, e.g., a mammal, e.g., amino acids 1-728 (SEQ ID NO: 2) of
human HGF.
[0056] The term "dHGF" refers to the deleted variant of HGF protein
produced by alternative splicing of the HGF gene in an animal,
e.g., a mammal, e.g., human HGF consisting of 723 amino acids (SEQ
ID NO: 3) with deletion of five amino acids in the 1st kringle
domain of the alpha chain (F, L, P, S and S) from the full length
HGF sequence.
[0057] The term "NK1" refers to an isoform of HGF from an animal,
e.g., a mammal, e.g., a human, consisting of the N-terminal hairpin
loop and kringle1 domains.
[0058] The term "NK2" refers to an isoform of HGF from an animal,
e.g., a mammal, e.g., a human, consisting of the N-terminal hairpin
loop, kringle1, and kringle2 domains.
[0059] The term "NK4" refers to an isoform of HGF from an animal,
e.g., a mammal, e.g., a human, consisting of the N-terminal hairpin
loop, kringle1, kringle2, kringle3, and kringle4 domains.
[0060] The structure and function of HGF has been extensively
studied and one of skill in the art is aware of the amino acids in
the HGF sequence that are important for retaining substantially all
of the biological activity of the protein and that are preferably
not changed or only conservatively changed in any sequence variant
of HGF. See, e.g., Hartmann et al., Proc. Natl. Acad. Sci. USA
89:11574 (1992); Lokker et al., EMBO J. 11:2503 (1992), Zhou et
al., Structure 6:109 (1998), Ultsch et al., Structure 6:1383
(1998), Shimizu et al., Biochem. Biophys. Res. Commun. 189:1329
(1992), Yoshiyama et al., Biochem. Biophys. Res. Commun. 175:660
(1991), each herein incorporated by reference in its entirety. For
example, it appears that the N-terminal hairpin loop and kringle1
domains are required for cell proliferation activity. Other amino
acids that are not critical to biological activity may be deleted
and/or substituted more freely. One of skill in the art can prepare
variants of HGF isoforms using routine mutagenesis techniques, such
as those described in the references cited above, and identify
variants retaining substantially all of the biological activity of
the HGF isoform.
[0061] An embodiment of the hybrid HGF gene of the present
invention comprising the inherent intron is 7113 bp long and has
the nucleotide sequence of SEQ ID NO: 7.
[0062] A hybrid HGF gene may comprise a fragment of inherent intron
optionally having a small recombinant sequence inserted thereinto
between exons 4 and 5 of HGF cDNA. Herein, such a hybrid HGF gene
comprising a fragment of inherent intron is designated "HGF-X".
Examples of hybrid HGF genes include HGF-X2 (SEQ ID NO: 13), HGF-X3
(SEQ ID NO: 14), HGF-X6 (SEQ ID NO: 8), HGF-X7 (SEQ ID NO: 9) and
HGF-X8 (SEQ ID NO: 10).
Administration and Methods of Treatment
[0063] As described above, HGF has various biological functions,
and based on these various functions, HGF, a gene encoding HGF, or
a variant thereof, may be developed as a therapeutic agent for
treating ischemic or liver diseases. In the present invention, an
HGF DNA formulation is administered after reconstitution of the
lyophilized DNA formulation.
[0064] The term "reconstituted" or "reconstitution" refers to the
restoration to the original form, e.g., by rehydration, of a
substance previously altered for preservation and storage, e.g.,
the restoration to a liquid state of a DNA plasmid formulation that
has been previously dried and stored. The lyophilized composition
of the present invention may be reconstituted in any aqueous
solution which produces a stable, mono-dispersed solution suitable
for administration. Such aqueous solutions include, but are not
limited to: sterile water, TE, PBS, Tris buffer or normal
saline.
[0065] The concentration of reconstituted lyophilized DNA in the
methods of the current invention is adjusted depending on many
factors, including the amount of a formulation to be delivered, the
age and weight of the subject, the delivery method and route and
the immunogenicity of the antigen being delivered.
[0066] The reconstituted lyophilized DNA formulation of the
invention may be administered orally or via parenteral routes such
as intravenous, intramuscular, intraendocardial, intramyocardial,
intrapericardial, intraventricular, intraarticular, intradermal,
intracerebral, intrarenal, intrahepatic, intrasplenic,
intralymphatic, subcutaneous, intraabdominal, intratesticular,
intraovarian, intrauterine, sternal, intratracheal, intraplueral,
intrathoracic, intradural, intraspinal, intramedullary, intramural,
intrascorionic and arterial injection or infusion, or topically
through rectal, intranasal, inhalational or intraocular
administration. In certain embodiments, the method of delivery is
intramuscular, intramyocardial, intravenous, intracerebral, or
intrarenal.
[0067] It should be understood that the typical daily dose of the
reconstituted lyophilized DNA formulation of the present invention
ought to be determined in light of various relevant factors
including the conditions to be treated, the chosen route of
administration, the age, sex and body weight of the individual
patient, and the severity of the patient's symptom, and can be
administrated in a single dose or in divided dose. Therefore, the
daily dose should not be construed as a limitation to the scope of
the invention in any way.
[0068] The term "treat," "treating," or "treatment" of an ischemic
or liver disease, as used herein, refers to the administration to a
subject of a factor, e.g. a HGF, e.g., a hybrid HGF, or variant
thereof, in an amount sufficient to result in amelioration of one
or more symptoms of the ischemic or liver disease, or prevent
advancement of the ischemic or liver disease.
[0069] An "ischemic disease" refers to a disease associated with a
deficient supply of blood to a body part (as the heart or brain)
that is due to obstruction of the inflow of arterial blood (as by
the narrowing of arteries by spasm or disease). Examples of
ischemic diseases include coronary artery disease (CAD) and
peripheral artery disease (PAD).
[0070] The term "liver disease" applies to many diseases and
disorders that cause the liver to function improperly or cease
functioning. HGF is a major agent promoting hepatocyte
proliferation, and acts in concert with transforming growth
factor-alpha and heparin-binding epidermal growth factor during
liver regeneration. Additionally, HGF ameliorates hepatic injury
via anti-apoptotic effects in animal models of fulminant hepatic
failure, and attenuates hepatic fibrosis in animals with liver
cirrhosis. Consequently, HGF is considered to not only induce liver
regeneration, but also to inhibit disease progression and
ameliorate hepatic fibrosis in patients suffering from intractable
liver diseases. With respect to the treatment of liver disease, the
reconstituted lyophilized DNA formulation of the invention may be
administered according to the delivery methods as set forth above.
In certain embodiments, the method of delivery in the treatment of
liver disease will be intravenous, intraarterial, or
intrahepatic.
[0071] In certain aspects of the invention, the reconstituted HGF
DNA formulation can comprise two or more isoforms of HGF. The HGF
isoforms may be previously lyophilized separately, or in the same
DNA formulation. Both of these lyophilized isoforms, after
reconstitution, can be administered separately or at the same time,
i.e., co-administered; separate reconstituted plasmid DNA
formulations for the two or more isoforms of HGF may be
administered or co-administered or a single expression plasmid
containing genes for two or more isoforms of HGF and capable of
expressing the genes for the two or more isoforms of HGF may be
administered. For example, the two isoforms flHGF and dHGF may be
administered using two separate plasmids. Alternatively, the two
separate plasmids containing genes for flHGF and dHGF may be used
for co-administration. Finally, a single expression plasmid
containing genes for both flHGF and dHGF may be administered. In
certain aspects of the invention, the flHGF and dHGF on a single
expression plasmid is encoded by the same polynucleotide or by
separate polynucleotides.
[0072] There are a number of approaches to include more than one
polynucleotide capable of expressing an HGF isoform on a single
plasmid. These include, for example, the use of Internal Ribosome
Entry Site (IRES) sequences, dual promoters/expression cassettes,
and fusion proteins. The two or more isoforms expressed from the
same plasmid or on two separate plasmids, as discussed above, are
selected from the group consisting of flHGF, dHGF, NK1, NK2, and
NK4 or selected from the group consisting of SEQ ID NOs: 2 to 6.
The two or more isoforms can also include additional HGF isoforms
known to one of ordinary skill in the art.
[0073] In certain aspects of the invention, the plasmid DNA is
administered through direct intracellular injection and, more
preferably, by the use of a syringe or a catheter. Catheters have
been used to introduce recombinant genes in vivo (see, e.g., E. G.
Nabel, et al., Proc. Natl. Acad. Sci. USA 89, 5157 (1992); E. G.
Nabel, et al., Science 249, 1285 (1990); E. G. Nabel, et al.,
Science 244, 1342 (1989); E. G. Nabel, et al., J. Clin. Invest. 91,
1822 (1993); G. Plautz, et al., Circ. 83, 578 (1991); E. G. Nabel,
et al., Nature (1993) (in press)). Utilization of a catheter
provides the ability to deliver the plasmid DNA into the cells
which are difficult to access by the use of a syringe.
[0074] The plasmid DNA can be administered through intraarterial or
intravenous injection and, more preferably, by the use of a syringe
or a catheter. For example, the femoral artery may be used to
deliver plasmid DNA to the heart; the portal vein may be used to
deliver plasmid DNA to the liver.
[0075] Administration of the plasmid DNA of the invention can also
be accomplished by gene transfer into target cells, in situ, to
optimize the subsequent delivery of genes in vivo.
[0076] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of cell biology, cell
culture, molecular biology (including PCR), vaccinology,
microbiology, recombinant DNA, and immunology, which are within the
skill of the art. Such techniques are explained fully in the
literature. See, for example, Molecular Cloning A Laboratory
Manual, 2nd Ed., Sambrook et al., ed., Cold Spring Harbor
Laboratory Press: (1989); DNA Cloning, Volumes I and II (D. N.
Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed.,
1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid
Hybridization (B. D. Hames & S. J. Higgins eds. 1984);
Transcription And Translation (B. D. Hames & S. J. Higgins eds.
1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc.,
1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal,
A Practical Guide To Molecular Cloning (1984); the treatise,
Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer
Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds.,
1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols.
154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And
Molecular Biology (Mayer and Walker, eds., Academic Press, London,
1987); and in Ausubel et al., Current Protocols in Molecular
Biology, John Wiley and Sons, Baltimore, Md. (1989). Each of the
references cited in this paragraph is incorporated herein by
reference in its entirety.
[0077] The following Examples are given for the purpose of
illustration only, and are not intended to limit the scope of the
invention.
EXAMPLES
Example 1
Preparation of Plasmid
[0078] The plasmid pCK-HGF-X7 (WO 03/078568) which is designed to
express hepatocyte growth factor (HGF) protein was used in the
experiment.
[0079] E. coli (TOP10, Invitrogen, USA) were transformed with
pCK-HGF-X7, and a single colony was isolated. The isolated colony
was then cultured in LB media containing 30 .mu.g/mL kanamycin.
Plasmid DNA was purified using an EndoFree plasmid Giga kit
(Qiagen, USA), and re-suspended in saline containing 0.9% NaCl at a
final DNA concentration of 1.0 to 2.0 mg/mL.
Example 2
Lyophilization
[0080] Formulations of pCK-HGF-X7 were prepared in saline
containing 0.9% NaCl at a final DNA concentration of 0.5 mg/mL or 1
mg/mL, with sucrose (0.25, 1.1, 5, 10 or 20% w/v) or mannitol (1.2,
4.85 or 10% w/v). Table 1A and 1B show the percentage sucrose and
mannitol, respectively, and the corresponding carbohydrate/DNA
(w/w) ratios for the tested pCK-HGF-X7 formulations.
TABLE-US-00001 TABLE 1A Percent Sucrose DNA Sucrose Sucrose Sucrose
(mg/ml) (%) (mg/ml)) to DNA ratio (w/w) 0.5 0.25 2.5 5 0.5 1.1 11
22 0.5 5 50 100 0.5 10 100 200 0.5 20 200 400 1 0.25 2.5 2.5 1 1.1
11 11 1 5 50 50 1 10 100 100 1 20 200 200
TABLE-US-00002 TABLE 1B Percent Mannitol DNA Mannitol Mannitol
Mannitol (mg/ml) (%) (mg/ml)) to DNA ratio (w/w) 0.5 1.2 12 24 0.5
4.85 48.5 97 0.5 10 100 200 1 1.2 12 12 1 4.85 48.5 48.5 1 10 100
100
[0081] The suspended plasmid DNA was then lyophilized with
Production-Master Freeze Dryer (C&H Cooling & Heating
Systems, Korea). The temperature was lowered to -50.degree. C. for
4 hours at 100 mTorr. Then, the temperature was raised to
-40.degree. C. for 12 hours, -30.degree. C. for 6 hours,
-20.degree. C. for 6 hours, -10.degree. C. for 6 hours, 0.degree.
C. for 6 hours, 10.degree. C. for 6 hours and 30.degree. C. for 24
hours, progressively, at 28.about.29 mTorr. The lyophilized plasmid
DNA was kept at -20.degree. C. until analyzed.
[0082] The suspended plasmid DNA was also lyophilized with
Production-Master Freeze Dryer (C&H Cooling & Heating
Systems, Korea). The temperature was lowered to 5.degree. C. for 1
minute, and -50.degree. C. for 2 hours at 100 mTorr. Then, the
temperature was raised to -40.degree. C. for 6 hours, -35.degree.
C. for 3 hours, -30.degree. C. for 6 hours, -25.degree. C. for 3
hours, -20.degree. C. for 3 hours, -15.degree. C. for 3 hours,
-10.degree. C. for 6 hours, -5.degree. C. for 3 hours, 0.degree. C.
for 6 hours, and 30.degree. C. for 17 hours, progressively, at
28.about.29 mTorr. The lyophilized plasmid DNA was kept at
-20.degree. C. until analyzed.
[0083] The suspended plasmid DNA was also lyophilized with
Production-Master Freeze Dryer (C&H Cooling & Heating
Systems, Korea). The temperature was lowered to 5.degree. C. for 1
minute, -10.degree. C. for 1 minute, -20.degree. C. for 1 minute,
-30.degree. C. for 1 minute, and -50.degree. C. for 1 minute at 150
mTorr. The temperature was maintained at -50.degree. C. for another
2 hours at 150 mTorr. Then, the temperature was raised to
-45.degree. C. for 6 hours, -40.degree. C. for 3 hours, -35.degree.
C. for 6 hours, -30.degree. C. for 3 hours, -25.degree. C. for 6
hours, -20.degree. C. for 3 hours, -15.degree. C. for 6 hours,
-10.degree. C. for 3 hours, -5.degree. C. for 6 hours, 0.degree. C.
for 12 hours, 10.degree. C. for 3 hours, 20.degree. C. for 6 hours,
and 30.degree. C. for 29 hours, progressively, at 30 mTorr. The
lyophilized plasmid DNA was kept at -20.degree. C. until
analyzed.
[0084] The lyophilized formulations prepared above were analyzed
for in vitro gene expression efficiency according to the methods
described in Example 3. The in vitro results for these preparations
were the same.
Example 3
Effects of Lyophilization on In Vitro Gene Expression Efficiency of
Plasmid DNA
[0085] 1. Materials and methods
[0086] To assess the effects of the lyophilization on gene
expression efficiency of plasmid DNA, the lyophilized plasmid DNA
was transfected into 293T cells, and the level of HGF expression
was measured. As a control, non-lyophilized plasmid DNA was also
transfected.
[0087] Four micrograms of pCK-HGF-X7 in various formulations (as
noted above in Example 1) were transfected into 1.times.10.sup.6
293T cells using FuGENE6 (Roche Diagnostics, Germany) (n=5). Before
transfection, 1 mg of the lyophilized plasmid DNA was reconstituted
with 2 ml of water for injection to the final concentration of 0.5
mg/mL.
[0088] Two days after transfection, the culture supernatants were
obtained and analyzed for HGF expression using a human HGF ELISA
kit (R&D Systems, MN, USA), according to the manufacturer's
recommendations. The ELISA results were statistically assessed by
Dunnett's multiple comparison test using SPSS program (version
13.0, SPSS. Inc, USA).
2. Results and Discussion
[0089] The results of HGF gene expression are provided in FIG. 1.
Contrary to previous reports, lyophilization did not affect the in
vitro gene expression efficiency of plasmid DNA. Among various
formulations, the HGF level from pCK-HGF-X7 lyophilized with 1.1%
sucrose and 0.9% NaCl was significantly higher than that from
non-lyophilized pCK-HGF-X7 (p=0.001) (FIG. 1).
[0090] These results indicate that the lyophilization formulation
containing 1.1% Sucrose and 0.9% NaCl would be more suitable for
pCK-HGF-X7 than a non-lyophilized formulation.
Example 4
Comparative Analysis of In Vivo Gene Expression Between
Non-Lyophilized and Lyophilized pCK-HGF-X7
1. Materials and Methods
[0091] Thirteen 5-week old BALB/c mice (males, Charles River) were
obtained for each group, and provided with food and water ad
libitum. The mice were allowed 7 days of rest before being
subjected to the experiment.
[0092] Mice were injected with 100 .mu.g of non-lyophilized
pCK-HGF-X7 containing 0.9% NaCl (NL-HGF-X7) or pCK-HGF-X7
lyophilized with 1.1% sucrose and 0.9% NaCl (L-HGF-X7) into the
tibialis cranialis, and were sacrificed at day 7 after treatment.
The lyophilized plasmid DNA was reconstituted with water to the
final concentration of 0.5 mg/mL before injection. To measure the
level of HGF protein expression, the injected muscles were
collected, and the muscle tissue was lysed with 500 .mu.L of cell
lysis buffer (50 mM NaCl, 0.2% sodium dodecyl sulfate, 0.5% sodium
deoxycholate, 2% IGEPAL CA-630, 25 mM Tris-HCl, pH7.4, 1 mM
phenylmethylsulfonyl fluoride) for 16 hours at 4.degree. C. The
lysates were centrifuged at 12,000 rpm for 5 minutes, and the
supernatants were harvested and analyzed for HGF expression using a
human HGF ELISA kit (R&D Systems).
[0093] The ELISA results were statistically assessed by one way
ANOVA and subsequent Tukey's Test using SPSS program (version
13.0).
2. Results and discussion
[0094] An average of 246 ng/mL of HGF protein was produced from the
animals administered with pCK-HGF-X7 lyophilized with 1.1% sucrose
and 0.9% NaCl (L-HGF-X7), while the animals administered with
non-lyophilized pCK-HGF-X7 expressed 76 ng/mL of HGF (FIG. 2). This
result indicates that pCK-HGF-X7 lyophilized with 1.1% sucrose and
0.9% NaCl can express HGF protein more efficiently than
non-lyophilized pCK-HGF-X7 (p<0.001).
Example 5
Comparative Analysis of Therapeutic Effects on Porcine Ischemic
Heart Disease Model Between Non-Lyophilized and Lyophilized
pCK-HGF-X7
[0095] 1. Materials and methods
(1) Animals
[0096] Eleven Yorkshire pigs (male, 28 to 30 kg, Clinical Research
Institute in Seoul National University Hospital) were obtained and
provided with food two-times per day and water ad libitum. The pigs
were allowed 7 days of rest before being subjected to the
experiment. The overall experimental plan is shown in FIG. 3.
(2) Establishment of the Porcine Ischemic Heart Disease Model
[0097] Xylazine (2 mg/kg), ketamine (20 mg/kg), and atropine (0.05
mg/kg) were injected intramuscularly into each pig. Twenty minutes
later, a 22-gauge Medicut sheath was inserted into the superficial
femoral artery for continuous monitoring of the blood pressure.
Thiopental sodium (10 mg/kg) was injected intravenously, and
endotracheal intubation was performed via the orotracheal route.
Anesthesia was maintained by inhalation of enflurane. During the
operation, positive pressure ventilation and an oxygen fraction of
30%.about.40% were maintained. Electrocardiograms, oxygen
saturation and arterial blood pressure were monitored
continuously.
[0098] Left thoracotomy was then performed. After opening the
pericardium followed by exploration of the left anterior descending
coronary artery (LAD), 2% lidocaine (1 mg/kg) was injected
intravenously and the distal one third of the LAD was ligated for 3
minutes, leaving the second diagonal branch as much as possible.
Reperfusion (ischemic preconditioning) was performed for 5 minutes
using 5-0 polypropylene sutures buttressed with a small piece of
Nelaton (4 Fr). After this single ischemic preconditioning, the
distal LAD was ligated and ST-segment depression or elevation on
the monitored electrocardiogram was confirmed. Additional lidocaine
(1 mg/kg) was injected intravenously 15 minutes after the ligation,
and the pericardium and thoracotomy wounds were closed. A single 28
Fr chest tube connected to wall suction was removed immediately
after enough spontaneous respiration returned, followed by the
removal of the endotracheal tube.
[0099] All protocols were approved by the Seoul National University
Animal Care and Use Committee.
(3) Intramyocardial Injection of Plasmids
[0100] Twenty eight days after the ligation of the coronary artery,
re-thoracotomy was performed. Using 27 gauge insulin injection
needles, a total dose of 1 mg of pCK-HGF-X7 lyophilized with 1.1%
sucrose and 0.9% NaCl (L-HGF-X7, n=3) or non-lyophilized pCK-HGF-X7
containing 0.9% NaCl (NL-HGF-X7, n=4) was injected into the
anterolateral ischemic border zone which lies between the fibrotic
infarction area and the grossly normal myocardium along the course
of the second diagonal branch. A total of five sites were injected.
Each site was injected with 0.2 mg of plasmid DNA and the interval
between injection sites was 1.5 cm. The lyophilized plasmid DNA was
reconstituted with water to the final concentration of 1 mg/mL
before injection. As a control, the identical amount of
non-lyophilized pCK containing 0.9% NaCl (n=4) was injected into
the anterolateral ischemic border zone. The injection points were
marked with suture tags using metal rings.
(4) Myocardial Single Photon Emission Computed Tomography
[0101] Twenty six days after the surgical induction of myocardial
infarction, .sup.99mTc-MIBI gated single photon emission computed
tomography (SPECT)(Vertex EPIC, ADAC Labs, CA., USA) was performed
to set a baseline before plasmid injection. The gated SPECT was
repeated 28 days later (on Day 54 after the induction of the
myocardial infarction).
[0102] A 20-segment model was chosen for a segmental analysis. Six
segments corresponding to the cardiac base were excluded from the
analysis because this region could be easily influenced by the
diaphragmatic attenuation or some artifacts around the heart; also
because the heart base was far away from the sites of the distal
coronary ligation and plasmid injection.
[0103] The SPECT images constructed by electrocardiography gating
were analyzed by an auto-quantitation program (AutoQUANT, ADAC
Labs, CA., USA), which is believed to eliminate the possible bias
by any associated technician's manipulation.
[0104] The amount of segmental perfusion was quantified by
measuring the uptake of .sup.99mTc-MIBI and calculated as a
percentage of the maximum uptake. When the segmental perfusion thus
estimated was less than 70%, it was defined as an underperfused
segment and used as the target of plasmid delivery. Segments
remaining well perfused even after the coronary ligation were also
excluded, as they would probably get no benefit from the
therapeutic angiogenesis. Wall thickening in the systolic phase was
indicated as a percentage of the end diastolic wall thickness on
the gated images.
(5) Statistics
[0105] Data are presented as the percent improvement compared to
the baseline. All data were analyzed using SPSS (version 13.0). The
statistical analysis of the myocardial perfusion and the segmental
wall-thickening was performed using paired-samples Student
t-test.
2. Results
[0106] Within each treatment group, the changes in the segmental
perfusion before and after the plasmid DNA injection were compared.
The baseline values for the average of segmental perfusion measured
on Day 26 after LAD ligation were 39.0.+-.14.6, 43.4.+-.13.4 and
36.9.+-.16.3% for pCK, NL-HGF-X7 and L-HGF-X7 treatment group,
respectively. .sup.99mTc-MIBI gated SPECT conducted on Day 54
showed that the average values of the segmental perfusion in the
pCK and NL-HGF-X7 groups were 37.8.+-.13.9% and 44.0.+-.14.5%,
respectively, which were not significantly different from the
baseline values measured on Day 26 (p=0.320 for pCK and 0.721 for
NL-HGF-X7). In contrast, the average value of the segmental
perfusion in the L-HGF-X7 treatment group was 41.2.+-.17.6%,
showing significant increase over the baseline value (p=0.003).
When the magnitude of the percent increase in the segmental
perfusion from baseline value was compared between groups, the
percent increase of the segmental perfusion in the L-HGF-X7
treatment group was 14.74% higher than that of pCK treatment group
(p=0.003), while the NL-HGF-X7 treatment group did not show
significant difference from the pCK treatment group (p=0.254) (FIG.
4).
[0107] In each treatment group, the changes in the segmental
wall-thickening before and after the DNA administration were also
compared. On Day 26, the average values of the segmental
wall-thickening were 24.7.+-.16.5, 33.4.+-.15.9 and 16.5.+-.15.9%
for pCK, NL-HGF-X7 and L-HGF-X7 treated group, respectively, and
there were no significant inter-group differences (p=NS). On Day
54, the average value of segmental wall-thickening for pCK,
NL-HGF-X7 and L-HGF-X7 treatment group was 27.9.+-.18.4,
43.1.+-.11.8, and 30.2.+-.10.7%, respectively. When the magnitude
of the percent increase in the segmental wall-thickening from
baseline value was compared between the treatment groups, the
percent increase in the L-HGF-X7 treatment group was 83.54%, which
was significantly higher than that of the NL-HGF-X7 group (28.99%)
(FIG. 5).
[0108] These results indicate that the intramyocardial
administration of the lyophilized formulation (L-HGF-X7) can more
efficiently increase the regional blood flow and wall-thickening in
the injected ischemic border area of left ventricle compared to the
non-lyophilized formulation (NL-HGF-X7). Without wishing to be
bound by theory, this is likely due to angiogenic and antifibrotic
activities of expressed HGF-X7.
3. Summary
[0109] The segmental perfusion and wall-thickening were
significantly increased in the lyophilized pCK-HGF-X7 treated group
as compared to those of the non-lyophilized pCK and pCK-HGF-X7
treated groups.
[0110] These results demonstrate that the intramyocardial
administration of pCK-HGF-X7 lyophilized with 1.1% sucrose and 0.9%
NaCl to the affected pigs could efficiently and stably increase the
regional perfusion and the wall-thickening in the ischemic
myocardium as compared to non-lyophilized pCK-HGF-X7.
[0111] While the invention has been described with respect to the
above specific embodiments, it should be recognized that various
modifications and changes may be made to the invention by those
skilled in the art which also fall within the scope of the
invention as defined by the appended claims.
Sequence CWU 1
1
1412187DNAHomo sapiens 1atgtgggtga ccaaactcct gccagccctg ctgctgcagc
atgtcctcct gcatctcctc 60ctgctcccca tcgccatccc ctatgcagag ggacaaagga
aaagaagaaa tacaattcat 120gaattcaaaa aatcagcaaa gactacccta
atcaaaatag atccagcact gaagataaaa 180accaaaaaag tgaatactgc
agaccaatgt gctaatagat gtactaggaa taaaggactt 240ccattcactt
gcaaggcttt tgtttttgat aaagcaagaa aacaatgcct ctggttcccc
300ttcaatagca tgtcaagtgg agtgaaaaaa gaatttggcc atgaatttga
cctctatgaa 360aacaaagact acattagaaa ctgcatcatt ggtaaaggac
gcagctacaa gggaacagta 420tctatcacta agagtggcat caaatgtcag
ccctggagtt ccatgatacc acacgaacac 480agctttttgc cttcgagcta
tcggggtaaa gacctacagg aaaactactg tcgaaatcct 540cgaggggaag
aagggggacc ctggtgtttc acaagcaatc cagaggtacg ctacgaagtc
600tgtgacattc ctcagtgttc agaagttgaa tgcatgacct gcaatgggga
gagttatcga 660ggtctcatgg atcatacaga atcaggcaag atttgtcagc
gctgggatca tcagacacca 720caccggcaca aattcttgcc tgaaagatat
cccgacaagg gctttgatga taattattgc 780cgcaatcccg atggccagcc
gaggccatgg tgctatactc ttgaccctca cacccgctgg 840gagtactgtg
caattaaaac atgcgctgac aatactatga atgacactga tgttcctttg
900gaaacaactg aatgcatcca aggtcaagga gaaggctaca ggggcactgt
caataccatt 960tggaatggaa ttccatgtca gcgttgggat tctcagtatc
ctcacgagca tgacatgact 1020cctgaaaatt tcaagtgcaa ggacctacga
gaaaattact gccgaaatcc agatgggtct 1080gaatcaccct ggtgttttac
cactgatcca aacatccgag ttggctactg ctcccaaatt 1140ccaaactgtg
atatgtcaca tggacaagat tgttatcgtg ggaatggcaa aaattatatg
1200ggcaacttat cccaaacaag atctggacta acatgttcaa tgtgggacaa
gaacatggaa 1260gacttacatc gtcatatctt ctgggaacca gatgcaagta
agctgaatga gaattactgc 1320cgaaatccag atgatgatgc tcatggaccc
tggtgctaca cgggaaatcc actcattcct 1380tgggattatt gccctatttc
tcgttgtgaa ggtgatacca cacctacaat agtcaattta 1440gaccatcccg
taatatcttg tgccaaaacg aaacaattgc gagttgtaaa tgggattcca
1500acacgaacaa acataggatg gatggttagt ttgagataca gaaataaaca
tatctgcgga 1560ggatcattga taaaggagag ttgggttctt actgcacgac
agtgtttccc ttctcgagac 1620ttgaaagatt atgaagcttg gcttggaatt
catgatgtcc acggaagagg agatgagaaa 1680tgcaaacagg ttctcaatgt
ttcccagctg gtatatggcc ctgaaggatc agatctggtt 1740ttaatgaagc
ttgccaggcc tgctgtcctg gatgattttg ttagtacgat tgatttacct
1800aattatggat gcacaattcc tgaaaagacc agttgcagtg tttatggctg
gggctacact 1860ggattgatca actatgatgg cctattacga gtggcacatc
tctatataat gggaaatgag 1920aaatgcagcc agcatcatcg agggaaggtg
actctgaatg agtctgaaat atgtgctggg 1980gctgaaaaga ttggatcagg
accatgtgag ggggattatg gtggcccact tgtttgtgag 2040caacataaaa
tgagaatggt tcttggtgtc attgttcctg gtcgtggatg tgccattcca
2100aatcgtcctg gtatttttgt ccgagtagca tattatgcaa aatggataca
caaaattatt 2160ttaacatata aggtaccaca gtcatag 21872728PRTHomo
sapiens 2Met Trp Val Thr Lys Leu Leu Pro Ala Leu Leu Leu Gln His
Val Leu1 5 10 15Leu His Leu Leu Leu Leu Pro Ile Ala Ile Pro Tyr Ala
Glu Gly Gln 20 25 30 Arg Lys Arg Arg Asn Thr Ile His Glu Phe Lys
Lys Ser Ala Lys Thr 35 40 45Thr Leu Ile Lys Ile Asp Pro Ala Leu Lys
Ile Lys Thr Lys Lys Val 50 55 60Asn Thr Ala Asp Gln Cys Ala Asn Arg
Cys Thr Arg Asn Lys Gly Leu65 70 75 80Pro Phe Thr Cys Lys Ala Phe
Val Phe Asp Lys Ala Arg Lys Gln Cys 85 90 95Leu Trp Phe Pro Phe Asn
Ser Met Ser Ser Gly Val Lys Lys Glu Phe 100 105 110 Gly His Glu Phe
Asp Leu Tyr Glu Asn Lys Asp Tyr Ile Arg Asn Cys 115 120 125Ile Ile
Gly Lys Gly Arg Ser Tyr Lys Gly Thr Val Ser Ile Thr Lys 130 135
140Ser Gly Ile Lys Cys Gln Pro Trp Ser Ser Met Ile Pro His Glu
His145 150 155 160Ser Phe Leu Pro Ser Ser Tyr Arg Gly Lys Asp Leu
Gln Glu Asn Tyr 165 170 175Cys Arg Asn Pro Arg Gly Glu Glu Gly Gly
Pro Trp Cys Phe Thr Ser 180 185 190 Asn Pro Glu Val Arg Tyr Glu Val
Cys Asp Ile Pro Gln Cys Ser Glu 195 200 205Val Glu Cys Met Thr Cys
Asn Gly Glu Ser Tyr Arg Gly Leu Met Asp 210 215 220His Thr Glu Ser
Gly Lys Ile Cys Gln Arg Trp Asp His Gln Thr Pro225 230 235 240His
Arg His Lys Phe Leu Pro Glu Arg Tyr Pro Asp Lys Gly Phe Asp 245 250
255Asp Asn Tyr Cys Arg Asn Pro Asp Gly Gln Pro Arg Pro Trp Cys Tyr
260 265 270 Thr Leu Asp Pro His Thr Arg Trp Glu Tyr Cys Ala Ile Lys
Thr Cys 275 280 285Ala Asp Asn Thr Met Asn Asp Thr Asp Val Pro Leu
Glu Thr Thr Glu 290 295 300Cys Ile Gln Gly Gln Gly Glu Gly Tyr Arg
Gly Thr Val Asn Thr Ile305 310 315 320Trp Asn Gly Ile Pro Cys Gln
Arg Trp Asp Ser Gln Tyr Pro His Glu 325 330 335His Asp Met Thr Pro
Glu Asn Phe Lys Cys Lys Asp Leu Arg Glu Asn 340 345 350 Tyr Cys Arg
Asn Pro Asp Gly Ser Glu Ser Pro Trp Cys Phe Thr Thr 355 360 365Asp
Pro Asn Ile Arg Val Gly Tyr Cys Ser Gln Ile Pro Asn Cys Asp 370 375
380Met Ser His Gly Gln Asp Cys Tyr Arg Gly Asn Gly Lys Asn Tyr
Met385 390 395 400Gly Asn Leu Ser Gln Thr Arg Ser Gly Leu Thr Cys
Ser Met Trp Asp 405 410 415Lys Asn Met Glu Asp Leu His Arg His Ile
Phe Trp Glu Pro Asp Ala 420 425 430 Ser Lys Leu Asn Glu Asn Tyr Cys
Arg Asn Pro Asp Asp Asp Ala His 435 440 445Gly Pro Trp Cys Tyr Thr
Gly Asn Pro Leu Ile Pro Trp Asp Tyr Cys 450 455 460Pro Ile Ser Arg
Cys Glu Gly Asp Thr Thr Pro Thr Ile Val Asn Leu465 470 475 480Asp
His Pro Val Ile Ser Cys Ala Lys Thr Lys Gln Leu Arg Val Val 485 490
495Asn Gly Ile Pro Thr Arg Thr Asn Ile Gly Trp Met Val Ser Leu Arg
500 505 510 Tyr Arg Asn Lys His Ile Cys Gly Gly Ser Leu Ile Lys Glu
Ser Trp 515 520 525Val Leu Thr Ala Arg Gln Cys Phe Pro Ser Arg Asp
Leu Lys Asp Tyr 530 535 540Glu Ala Trp Leu Gly Ile His Asp Val His
Gly Arg Gly Asp Glu Lys545 550 555 560Cys Lys Gln Val Leu Asn Val
Ser Gln Leu Val Tyr Gly Pro Glu Gly 565 570 575Ser Asp Leu Val Leu
Met Lys Leu Ala Arg Pro Ala Val Leu Asp Asp 580 585 590 Phe Val Ser
Thr Ile Asp Leu Pro Asn Tyr Gly Cys Thr Ile Pro Glu 595 600 605Lys
Thr Ser Cys Ser Val Tyr Gly Trp Gly Tyr Thr Gly Leu Ile Asn 610 615
620Tyr Asp Gly Leu Leu Arg Val Ala His Leu Tyr Ile Met Gly Asn
Glu625 630 635 640Lys Cys Ser Gln His His Arg Gly Lys Val Thr Leu
Asn Glu Ser Glu 645 650 655Ile Cys Ala Gly Ala Glu Lys Ile Gly Ser
Gly Pro Cys Glu Gly Asp 660 665 670 Tyr Gly Gly Pro Leu Val Cys Glu
Gln His Lys Met Arg Met Val Leu 675 680 685Gly Val Ile Val Pro Gly
Arg Gly Cys Ala Ile Pro Asn Arg Pro Gly 690 695 700Ile Phe Val Arg
Val Ala Tyr Tyr Ala Lys Trp Ile His Lys Ile Ile705 710 715 720Leu
Thr Tyr Lys Val Pro Gln Ser 7253723PRTHomo sapiens 3Met Trp Val Thr
Lys Leu Leu Pro Ala Leu Leu Leu Gln His Val Leu1 5 10 15Leu His Leu
Leu Leu Leu Pro Ile Ala Ile Pro Tyr Ala Glu Gly Gln 20 25 30Arg Lys
Arg Arg Asn Thr Ile His Glu Phe Lys Lys Ser Ala Lys Thr 35 40 45Thr
Leu Ile Lys Ile Asp Pro Ala Leu Lys Ile Lys Thr Lys Lys Val 50 55
60Asn Thr Ala Asp Gln Cys Ala Asn Arg Cys Thr Arg Asn Lys Gly Leu65
70 75 80Pro Phe Thr Cys Lys Ala Phe Val Phe Asp Lys Ala Arg Lys Gln
Cys 85 90 95Leu Trp Phe Pro Phe Asn Ser Met Ser Ser Gly Val Lys Lys
Glu Phe 100 105 110 Gly His Glu Phe Asp Leu Tyr Glu Asn Lys Asp Tyr
Ile Arg Asn Cys 115 120 125Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gly
Thr Val Ser Ile Thr Lys 130 135 140Ser Gly Ile Lys Cys Gln Pro Trp
Ser Ser Met Ile Pro His Glu His145 150 155 160Ser Tyr Arg Gly Lys
Asp Leu Gln Glu Asn Tyr Cys Arg Asn Pro Arg 165 170 175Gly Glu Glu
Gly Gly Pro Trp Cys Phe Thr Ser Asn Pro Glu Val Arg 180 185 190 Tyr
Glu Val Cys Asp Ile Pro Gln Cys Ser Glu Val Glu Cys Met Thr 195 200
205Cys Asn Gly Glu Ser Tyr Arg Gly Leu Met Asp His Thr Glu Ser Gly
210 215 220Lys Ile Cys Gln Arg Trp Asp His Gln Thr Pro His Arg His
Lys Phe225 230 235 240Leu Pro Glu Arg Tyr Pro Asp Lys Gly Phe Asp
Asp Asn Tyr Cys Arg 245 250 255Asn Pro Asp Gly Gln Pro Arg Pro Trp
Cys Tyr Thr Leu Asp Pro His 260 265 270 Thr Arg Trp Glu Tyr Cys Ala
Ile Lys Thr Cys Ala Asp Asn Thr Met 275 280 285Asn Asp Thr Asp Val
Pro Leu Glu Thr Thr Glu Cys Ile Gln Gly Gln 290 295 300Gly Glu Gly
Tyr Arg Gly Thr Val Asn Thr Ile Trp Asn Gly Ile Pro305 310 315
320Cys Gln Arg Trp Asp Ser Gln Tyr Pro His Glu His Asp Met Thr Pro
325 330 335Glu Asn Phe Lys Cys Lys Asp Leu Arg Glu Asn Tyr Cys Arg
Asn Pro 340 345 350 Asp Gly Ser Glu Ser Pro Trp Cys Phe Thr Thr Asp
Pro Asn Ile Arg 355 360 365Val Gly Tyr Cys Ser Gln Ile Pro Asn Cys
Asp Met Ser His Gly Gln 370 375 380Asp Cys Tyr Arg Gly Asn Gly Lys
Asn Tyr Met Gly Asn Leu Ser Gln385 390 395 400Thr Arg Ser Gly Leu
Thr Cys Ser Met Trp Asp Lys Asn Met Glu Asp 405 410 415Leu His Arg
His Ile Phe Trp Glu Pro Asp Ala Ser Lys Leu Asn Glu 420 425 430 Asn
Tyr Cys Arg Asn Pro Asp Asp Asp Ala His Gly Pro Trp Cys Tyr 435 440
445Thr Gly Asn Pro Leu Ile Pro Trp Asp Tyr Cys Pro Ile Ser Arg Cys
450 455 460Glu Gly Asp Thr Thr Pro Thr Ile Val Asn Leu Asp His Pro
Val Ile465 470 475 480Ser Cys Ala Lys Thr Lys Gln Leu Arg Val Val
Asn Gly Ile Pro Thr 485 490 495Arg Thr Asn Ile Gly Trp Met Val Ser
Leu Arg Tyr Arg Asn Lys His 500 505 510 Ile Cys Gly Gly Ser Leu Ile
Lys Glu Ser Trp Val Leu Thr Ala Arg 515 520 525Gln Cys Phe Pro Ser
Arg Asp Leu Lys Asp Tyr Glu Ala Trp Leu Gly 530 535 540Ile His Asp
Val His Gly Arg Gly Asp Glu Lys Cys Lys Gln Val Leu545 550 555
560Asn Val Ser Gln Leu Val Tyr Gly Pro Glu Gly Ser Asp Leu Val Leu
565 570 575Met Lys Leu Ala Arg Pro Ala Val Leu Asp Asp Phe Val Ser
Thr Ile 580 585 590 Asp Leu Pro Asn Tyr Gly Cys Thr Ile Pro Glu Lys
Thr Ser Cys Ser 595 600 605Val Tyr Gly Trp Gly Tyr Thr Gly Leu Ile
Asn Tyr Asp Gly Leu Leu 610 615 620Arg Val Ala His Leu Tyr Ile Met
Gly Asn Glu Lys Cys Ser Gln His625 630 635 640His Arg Gly Lys Val
Thr Leu Asn Glu Ser Glu Ile Cys Ala Gly Ala 645 650 655Glu Lys Ile
Gly Ser Gly Pro Cys Glu Gly Asp Tyr Gly Gly Pro Leu 660 665 670 Val
Cys Glu Gln His Lys Met Arg Met Val Leu Gly Val Ile Val Pro 675 680
685Gly Arg Gly Cys Ala Ile Pro Asn Arg Pro Gly Ile Phe Val Arg Val
690 695 700Ala Tyr Tyr Ala Lys Trp Ile His Lys Ile Ile Leu Thr Tyr
Lys Val705 710 715 720Pro Gln Ser4207PRTHomo sapiens 4Met Trp Val
Thr Lys Leu Leu Pro Ala Leu Leu Leu Gln His Val Leu1 5 10 15Leu His
Leu Leu Leu Leu Pro Ile Ala Ile Pro Tyr Ala Glu Gly Gln 20 25 30Arg
Lys Arg Arg Asn Thr Ile His Glu Phe Lys Lys Ser Ala Lys Thr 35 40
45Thr Leu Ile Lys Ile Asp Pro Ala Leu Lys Ile Lys Thr Lys Lys Val
50 55 60Asn Thr Ala Asp Gln Cys Ala Asn Arg Cys Thr Arg Asn Lys Gly
Leu65 70 75 80Pro Phe Thr Cys Lys Ala Phe Val Phe Asp Lys Ala Arg
Lys Gln Cys 85 90 95Leu Trp Phe Pro Phe Asn Ser Met Ser Ser Gly Val
Lys Lys Glu Phe 100 105 110 Gly His Glu Phe Asp Leu Tyr Glu Asn Lys
Asp Tyr Ile Arg Asn Cys 115 120 125Ile Ile Gly Lys Gly Arg Ser Tyr
Lys Gly Thr Val Ser Ile Thr Lys 130 135 140Ser Gly Ile Lys Cys Gln
Pro Trp Ser Ser Met Ile Pro His Glu His145 150 155 160Ser Phe Leu
Pro Ser Ser Tyr Arg Gly Lys Asp Leu Gln Glu Asn Tyr 165 170 175Cys
Arg Asn Pro Arg Gly Glu Glu Gly Gly Pro Trp Cys Phe Thr Ser 180 185
190 Asn Pro Glu Val Arg Tyr Glu Val Cys Asp Ile Pro Gln Cys Ser 195
200 2055290PRTHomo sapiens 5Met Trp Val Thr Lys Leu Leu Pro Ala Leu
Leu Leu Gln His Val Leu1 5 10 15Leu His Leu Leu Leu Leu Pro Ile Ala
Ile Pro Tyr Ala Glu Gly Gln 20 25 30Arg Lys Arg Arg Asn Thr Ile His
Glu Phe Lys Lys Ser Ala Lys Thr 35 40 45Thr Leu Ile Lys Ile Asp Pro
Ala Leu Lys Ile Lys Thr Lys Lys Val 50 55 60Asn Thr Ala Asp Gln Cys
Ala Asn Arg Cys Thr Arg Asn Lys Gly Leu65 70 75 80Pro Phe Thr Cys
Lys Ala Phe Val Phe Asp Lys Ala Arg Lys Gln Cys 85 90 95Leu Trp Phe
Pro Phe Asn Ser Met Ser Ser Gly Val Lys Lys Glu Phe 100 105 110 Gly
His Glu Phe Asp Leu Tyr Glu Asn Lys Asp Tyr Ile Arg Asn Cys 115 120
125Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gly Thr Val Ser Ile Thr Lys
130 135 140Ser Gly Ile Lys Cys Gln Pro Trp Ser Ser Met Ile Pro His
Glu His145 150 155 160Ser Phe Leu Pro Ser Ser Tyr Arg Gly Lys Asp
Leu Gln Glu Asn Tyr 165 170 175Cys Arg Asn Pro Arg Gly Glu Glu Gly
Gly Pro Trp Cys Phe Thr Ser 180 185 190 Asn Pro Glu Val Arg Tyr Glu
Val Cys Asp Ile Pro Gln Cys Ser Glu 195 200 205Val Glu Cys Met Thr
Cys Asn Gly Glu Ser Tyr Arg Gly Leu Met Asp 210 215 220His Thr Glu
Ser Gly Lys Ile Cys Gln Arg Trp Asp His Gln Thr Pro225 230 235
240His Arg His Lys Phe Leu Pro Glu Arg Tyr Pro Asp Lys Gly Phe Asp
245 250 255Asp Asn Tyr Cys Arg Asn Pro Asp Gly Gln Pro Arg Pro Trp
Cys Tyr 260 265 270 Thr Leu Asp Pro His Thr Arg Trp Glu Tyr Cys Ala
Ile Lys Thr Cys 275 280 285Glu Thr 2906470PRTHomo sapiens 6Met Trp
Val Thr Lys Leu Leu Pro Ala Leu Leu Leu Gln His Val Leu1 5 10 15Leu
His Leu Leu Leu Leu Pro Ile Ala Ile Pro Tyr Ala Glu Gly Gln 20 25
30Arg Lys Arg Arg Asn Thr Ile His Glu Phe Lys Lys Ser Ala Lys Thr
35 40 45Thr Leu Ile Lys Ile Asp Pro Ala Leu Lys Ile Lys Thr Lys Lys
Val 50 55 60Asn Thr Ala Asp Gln Cys Ala Asn Arg Cys Thr Arg Asn Lys
Gly Leu65 70 75 80Pro Phe Thr Cys Lys Ala Phe Val Phe Asp Lys Ala
Arg Lys Gln Cys 85 90 95Leu Trp Phe Pro Phe Asn Ser Met Ser Ser Gly
Val Lys Lys Glu Phe 100 105 110 Gly His Glu Phe Asp Leu Tyr Glu Asn
Lys Asp Tyr Ile Arg Asn Cys 115 120 125Ile Ile Gly Lys Gly Arg Ser
Tyr Lys Gly Thr Val Ser Ile Thr Lys 130 135 140Ser Gly Ile Lys
Cys
Gln Pro Trp Ser Ser Met Ile Pro His Glu His145 150 155 160Ser Phe
Leu Pro Ser Ser Tyr Arg Gly Lys Asp Leu Gln Glu Asn Tyr 165 170
175Cys Arg Asn Pro Arg Gly Glu Glu Gly Gly Pro Trp Cys Phe Thr Ser
180 185 190 Asn Pro Glu Val Arg Tyr Glu Val Cys Asp Ile Pro Gln Cys
Ser Glu 195 200 205Val Glu Cys Met Thr Cys Asn Gly Glu Ser Tyr Arg
Gly Leu Met Asp 210 215 220His Thr Glu Ser Gly Lys Ile Cys Gln Arg
Trp Asp His Gln Thr Pro225 230 235 240His Arg His Lys Phe Leu Pro
Glu Arg Tyr Pro Asp Lys Gly Phe Asp 245 250 255Asp Asn Tyr Cys Arg
Asn Pro Asp Gly Gln Pro Arg Pro Trp Cys Tyr 260 265 270 Thr Leu Asp
Pro His Thr Arg Trp Glu Tyr Cys Ala Ile Lys Thr Cys 275 280 285Ala
Asp Asn Thr Met Asn Asp Thr Asp Val Pro Leu Glu Thr Thr Glu 290 295
300Cys Ile Gln Gly Gln Gly Glu Gly Tyr Arg Gly Thr Val Asn Thr
Ile305 310 315 320Trp Asn Gly Ile Pro Cys Gln Arg Trp Asp Ser Gln
Tyr Pro His Glu 325 330 335His Asp Met Thr Pro Glu Asn Phe Lys Cys
Lys Asp Leu Arg Glu Asn 340 345 350 Tyr Cys Arg Asn Pro Asp Gly Ser
Glu Ser Pro Trp Cys Phe Thr Thr 355 360 365Asp Pro Asn Ile Arg Val
Gly Tyr Cys Ser Gln Ile Pro Asn Cys Asp 370 375 380Met Ser His Gly
Gln Asp Cys Tyr Arg Gly Asn Gly Lys Asn Tyr Met385 390 395 400Gly
Asn Leu Ser Gln Thr Arg Ser Gly Leu Thr Cys Ser Met Trp Asp 405 410
415Lys Asn Met Glu Asp Leu His Arg His Ile Phe Trp Glu Pro Asp Ala
420 425 430 Ser Lys Leu Asn Glu Asn Tyr Cys Arg Asn Pro Asp Asp Asp
Ala His 435 440 445Gly Pro Trp Cys Tyr Thr Gly Asn Pro Leu Ile Pro
Trp Asp Tyr Cys 450 455 460Pro Ile Ser Arg Cys Glu465
47077113DNAArtificial Sequencesynthetic hepatocyte growth factor
hybrid 7atgtgggtga ccaaactcct gccagccctg ctgctgcagc atgtcctcct
gcatctcctc 60ctgctcccca tcgccatccc ctatgcagag ggacaaagga aaagaagaaa
tacaattcat 120gaattcaaaa aatcagcaaa gactacccta atcaaaatag
atccagcact gaagataaaa 180accaaaaaag tgaatactgc agaccaatgt
gctaatagat gtactaggaa taaaggactt 240ccattcactt gcaaggcttt
tgtttttgat aaagcaagaa aacaatgcct ctggttcccc 300ttcaatagca
tgtcaagtgg agtgaaaaaa gaatttggcc atgaatttga cctctatgaa
360aacaaagact acattagaaa ctgcatcatt ggtaaaggac gcagctacaa
gggaacagta 420tctatcacta agagtggcat caaatgtcag ccctggagtt
ccatgatacc acacgaacac 480aggtaagaac agtatgaaga aaagagatga
agcctctgtc ttttttacat gttaacagtc 540tcatattagt ccttcagaat
aattctacaa tcctaaaata acttagccaa cttgctgaat 600tgtattacgg
caaggtttat atgaattcat gactgatatt tagcaaatga ttaattaata
660tgttaataaa atgtagccaa aacaatatct taccttaatg cctcaatttg
tagatctcgg 720tatttgtgaa ataataacgt aaacttcgtt taaaaggatt
cttcttcctg tctttgagaa 780agtacggcac tgtgcagggg gagaggttga
ttgtgaaaaa tcagaggtag atgagaatct 840tactgagggc tgagggttct
ttaaccttgg tggatctcaa cattggttgc acattaaaat 900cacctgctgc
aagcccttga cgaatcttac ttagaagatg acaacacaga acaattaaat
960cagaatctct ggggagaata gggcaccagt attttttgag ctcccaccat
gattccaaag 1020tgcagccaaa tttgagaacc actgctaaaa gctcaagctt
cagattgacc agcttttcca 1080tctcacctat cgcctaaaga ccaaattgga
taaatgtgtt cattacgaca gatgggtact 1140atttaaagat gagtaaacac
aatatactta ggctcgtcag actgagagtt ttaatcatca 1200ctgaggaaaa
acatagatat ctaatactga ctggagtatt agtcaaggct tatttcacac
1260acaattttat cagaaaccaa agtagtttaa aacagctctc cccttattag
taatgcattg 1320gagggtttac tttaccatgt accttgctga gcactgtacc
ttgttaatct catttacttg 1380taatgagaac cacacagcgg gtagttttat
tggttctatt ttacctacat gacaaaactg 1440aagcataaaa acacttagta
agttttcagt gtcatgcaca actaggaagt gacatggcca 1500gaatataagc
ccagtcacca tcactctata acctgcgctt ttaacaactt cagggcatga
1560cacatttggc cggtcagtag aacccatgct gtgatttgtt tttgcagtgg
tggtgatgac 1620tgccttgttg aatccacttt ttattctatt ccattttggg
gacacaattc tgcaagatga 1680ttcttcatta ggaaacagag atgagttatt
gaccaacaca gaaagaaaaa gagtttgttg 1740ctccacactg ggattaaacc
tatgatcttg gcctaattaa cactagctag taagtgtcca 1800agctgatcat
ctctacaaca tttcaataac agaaaacaac aattttcaaa attagttact
1860tacaattatg tagaaatgcc tctaaaacac agtattttcc ttatattaca
aaaacaaaaa 1920ttataattgg ttttgtcctc ttttgagagt ttgcatggtg
ttactccctg catagtgaag 1980aaaacatttt atttaagtag atggatctaa
gtttttcatg aacaaaggaa tgacatttga 2040aatcaatcct accctagtcc
aggagaatgc attagattaa cctagtagag gtcttatttc 2100accctgagtt
ttctatgatc gtgattctct gctggaggag taattgtgaa atagatctct
2160ctgggaactg gcttcctagt ccaatcagct cttttaccaa tgaacacttc
cttgtgatat 2220agatgtttat ggccgagagg atccagtata ttaataaaat
ccctttttgt attcaatgag 2280ggaaacacat aattttcatc aattagcagc
ttattggaat atctgcatga tggtttaaca 2340cttttaagtg ttgactaaag
attaatttta cagaaaatag aaaaagaaat atgtttctgt 2400ctggaggaat
gatttattgt tgacccctaa attgaaatat tttactagtg gcttaatgga
2460aagatgatga aagatgatga aattaatgta gaagcttaac tagaaaatca
ggtgacctga 2520tatctacatc tgtatccttc attggccacc cagcattcat
taatgaatca gatgatggaa 2580tagatcaagt ttcctaggaa cacagtgaat
attaaaagaa aacaaaggga gcctagcacc 2640tagaagacct agtttatatt
tcaaagtata tttggatgta acccaatttt aaacatttcc 2700tcacttgtct
ctcttaaagc cttgccaaca gcaaggacag agaaccaaaa atagtgtata
2760tatgaataaa tgcttattac agaatctgct gactggcaca tgctttgtgt
gtaatgggtt 2820ctcataaaca cttgttgaat gaacacacat aagtgaaaga
gcatggctag gcttcatccc 2880ttggtcaaat atggggtgct aaagaaaagc
aggggaaata cattgggaca ctaacaaaaa 2940aaaacagtta atttaggtaa
aagataaaat acaccacaga atgaagaaaa gagatgaccc 3000agactgctct
ttaaccttca tgtcctagag aggtttttga tatgaattgc attcagaatt
3060gtggaaagga gcccatcttt tctcttcatt ttgattttat taactccaat
gggggaattt 3120tattcgtgtt ttggccatat ctacttttga tttctacatt
attctctctt cctttctacc 3180tgtatttgtc ctaataaatt gttgacttat
taattcacta cttcctcaca gctttttttt 3240ggctttacaa atccactgga
aaggtatatg ggtgtatcac tttgtgtatt tcggtgtgca 3300tgtgtagagg
ggacaaaaat cctctctcaa actataaata ttgagtattt gtgtattgaa
3360catttgctat aactactagg tttcttaaat aatcttaata tataaaatga
tatagaaaaa 3420gggaaattat agttcgtatt attcatctaa gtgaagagat
taaaacccag ggagtaaata 3480aattgtctaa ggactaaggt tgtatactat
ttaggtgata gatatggggc aaccgtatgg 3540gttttatgat taacaaataa
acttctcacc actctaccat atcaactttt ccataaaaga 3600gagctatagt
attctttgct taaataaatt tgattagtgc atgacttctt gaaaacatat
3660aaagcaaaag tcacatttga ttctatcaga aaagtgagta agccatggcc
caaacaaaag 3720atgcattaaa atattctgga atgatggagc taaaagtaag
aaaaatgact ttttaaaaaa 3780gtttactgtt aggaattgtg aaattatgct
gaattttagt tgcattataa tttttgtcag 3840tcatacggtc tgacaacctg
tcttatttct atttccccat atgaggaatg ctagttaagt 3900atggatatta
actattacta cttagatgca ttgaagttgc ataatatgga taatacttca
3960ctggttccct gaaaatgttt agttagtaat aagtctctta cactatttgt
tttgtccaat 4020aatttatatt ttctgaagac ttaactctag aatacactca
tgtcaaaatg aaagaatttc 4080attgcaaaat attgcttggt acatgacgca
tacctgtatt tgttttgtgt cacaacatga 4140aaaatgatgg tttattagaa
gtttcattgg gtaggaaaca catttgaatg gtatttacta 4200agatactaaa
atccttggac ttcactctaa ttttagtgcc atttagaact caaggtctca
4260gtaaaagtag aaataaagcc tgttaacaaa acacaagctg aatattaaaa
atgtaactgg 4320attttcaaag aaatgtttac tggtattacc tgtagatgta
tattctttat tatgatcttt 4380tgtgtaaagt ctggcagaca aatgcaatat
ctaattgttg agtccaatat cacaagcagt 4440acaaaagtat aaaaaagact
tggccttttc taatgtgtta aaatacttta tgctggtaat 4500aacactaaga
gtagggcact agaaatttta agtgaagata atgtgttgca gttactgcac
4560tcaatggctt actattataa accaaaactg ggatcactaa gctccagtca
gtcaaaatga 4620tcaaaattat tgaagagaat aagcaattct gttctttatt
aggacacagt agatacagac 4680tacaaagtgg agtgtgctta ataagaggta
gcatttgtta agtgtcaatt actctattat 4740cccttggagc ttctcaaaat
aaccatataa ggtgtaagat gttaaaggtt atggttacac 4800tcagtgcaca
ggtaagctaa taggctgaga gaagctaaat tacttactgg ggtctcacag
4860taagaaagtg agctgaagtt tcagcccaga tttaactgga ttctgggctc
tttattcatg 4920ttacttcatg aatctgtttc tcaattgtgc agaaaaaagg
gggctattta taagaaaagc 4980aataaacaaa caagtaatga tctcaaataa
gtaatgcaag aaatagtgag atttcaaaat 5040cagtggcagc gatttctcag
ttctgtccta agtggccttg ctcaatcacc tgctatcttt 5100tagtggagct
ttgaaattat gtttcagaca acttcgattc agttctagaa tgtttgactc
5160agcaaattca caggctcatc tttctaactt gatggtgaat atggaaattc
agctaaatgg 5220atgttaataa aattcaaacg ttttaaggac agatgaaaat
gacagaattt taaggtaaaa 5280tatatgaagg aatataagat aaaggatttt
tctaccttca gcaaaaacat acccactaat 5340tagtaaaatt aataggcaaa
aaaaagttgc atgctcttat actgtaatga ttatcatttt 5400aaaactagct
ttttgccttc gagctatcgg ggtaaagacc tacaggaaaa ctactgtcga
5460aatcctcgag gggaagaagg gggaccctgg tgtttcacaa gcaatccaga
ggtacgctac 5520gaagtctgtg acattcctca gtgttcagaa gttgaatgca
tgacctgcaa tggggagagt 5580tatcgaggtc tcatggatca tacagaatca
ggcaagattt gtcagcgctg ggatcatcag 5640acaccacacc ggcacaaatt
cttgcctgaa agatatcccg acaagggctt tgatgataat 5700tattgccgca
atcccgatgg ccagccgagg ccatggtgct atactcttga ccctcacacc
5760cgctgggagt actgtgcaat taaaacatgc gctgacaata ctatgaatga
cactgatgtt 5820cctttggaaa caactgaatg catccaaggt caaggagaag
gctacagggg cactgtcaat 5880accatttgga atggaattcc atgtcagcgt
tgggattctc agtatcctca cgagcatgac 5940atgactcctg aaaatttcaa
gtgcaaggac ctacgagaaa attactgccg aaatccagat 6000gggtctgaat
caccctggtg ttttaccact gatccaaaca tccgagttgg ctactgctcc
6060caaattccaa actgtgatat gtcacatgga caagattgtt atcgtgggaa
tggcaaaaat 6120tatatgggca acttatccca aacaagatct ggactaacat
gttcaatgtg ggacaagaac 6180atggaagact tacatcgtca tatcttctgg
gaaccagatg caagtaagct gaatgagaat 6240tactgccgaa atccagatga
tgatgctcat ggaccctggt gctacacggg aaatccactc 6300attccttggg
attattgccc tatttctcgt tgtgaaggtg ataccacacc tacaatagtc
6360aatttagacc atcccgtaat atcttgtgcc aaaacgaaac aattgcgagt
tgtaaatggg 6420attccaacac gaacaaacat aggatggatg gttagtttga
gatacagaaa taaacatatc 6480tgcggaggat cattgataaa ggagagttgg
gttcttactg cacgacagtg tttcccttct 6540cgagacttga aagattatga
agcttggctt ggaattcatg atgtccacgg aagaggagat 6600gagaaatgca
aacaggttct caatgtttcc cagctggtat atggccctga aggatcagat
6660ctggttttaa tgaagcttgc caggcctgct gtcctggatg attttgttag
tacgattgat 6720ttacctaatt atggatgcac aattcctgaa aagaccagtt
gcagtgttta tggctggggc 6780tacactggat tgatcaacta tgatggccta
ttacgagtgg cacatctcta tataatggga 6840aatgagaaat gcagccagca
tcatcgaggg aaggtgactc tgaatgagtc tgaaatatgt 6900gctggggctg
aaaagattgg atcaggacca tgtgaggggg attatggtgg cccacttgtt
6960tgtgagcaac ataaaatgag aatggttctt ggtgtcattg ttcctggtcg
tggatgtgcc 7020attccaaatc gtcctggtat ttttgtccga gtagcatatt
atgcaaaatg gatacacaaa 7080attattttaa catataaggt accacagtca tag
711384679DNAArtificial SequenceSynthetic HGF-X6 gene 8atgtgggtga
ccaaactcct gccagccctg ctgctgcagc atgtcctcct gcatctcctc 60ctgctcccca
tcgccatccc ctatgcagag ggacaaagga aaagaagaaa tacaattcat
120gaattcaaaa aatcagcaaa gactacccta atcaaaatag atccagcact
gaagataaaa 180accaaaaaag tgaatactgc agaccaatgt gctaatagat
gtactaggaa taaaggactt 240ccattcactt gcaaggcttt tgtttttgat
aaagcaagaa aacaatgcct ctggttcccc 300ttcaatagca tgtcaagtgg
agtgaaaaaa gaatttggcc atgaatttga cctctatgaa 360aacaaagact
acattagaaa ctgcatcatc ggtaaaggac gcagctacaa gggaacagta
420tctatcacta agagtggcat caaatgtcag ccctggagtt ccatgatacc
acacgaacac 480aggtaagaac agtatgaaga aaagagatga agcctctgtc
ttttttacat gttaacagtc 540tcatattagt ccttcagaat aattctacaa
tcctaaaata acttagccaa cttgctgaat 600tgtattacgg caaggtttat
atgaattcat gactgatatt tagcaaatga ttaattaata 660tgttaataaa
atgtagccaa aacaatatct taccttaatg cctcaatttg tagatctcgg
720tatttgtgga tcccttcctt tctacctgta tttgtcctaa taaattgttg
acttattaat 780tcactacttc ctcacagctt ttttttggct ttacaaatcc
actggaaagg tatatgggtg 840tatcactttg tgtatttcgg tgtgcatgtg
tagaggggac aaaaatcctc tctcaaacta 900taaatattga gtatttgtgt
attgaacatt tgctataact actaggtttc ttaaataatc 960ttaatatata
aaatgatata gaaaaaggga aattatagtt cgtattattc atctaagtga
1020agagattaaa acccagggag taaataaatt gtctaaggac taaggttgta
tactatttag 1080gtgatagata tggggcaacc gtatgggttt tatgattaac
aaataaactt ctcaccactc 1140taccatatca acttttccat aaaagagagc
tatagtattc tttgcttaaa taaatttgat 1200tagtgcatga cttcttgaaa
acatataaag caaaagtcac atttgattct atcagaaaag 1260tgagtaagcc
atggcccaaa caaaagatgc attaaaatat tctggaatga tggagctaaa
1320agtaagaaaa atgacttttt aaaaaagttt actgttagga attgtgaaat
tatgctgaat 1380tttagttgca ttataatttt tgtcagtcat acggtctgac
aacctgtctt atttctattt 1440ccccatatga ggaatgctag ttaagtatgg
atattaacta ttactactta gatgcattga 1500agttgcataa tatggataat
acttcactgg ttccctgaaa atgtttagtt agtaataagt 1560ctcttacact
atttgttttg tccaataatt tatattttct gaagacttaa ctctagaata
1620cactcatgtc aaaatgaaag aatttcattg caaaatattg cttggtacat
gacgcatacc 1680tgtatttgtt ttgtgtcaca acatgaaaaa tgatggttta
ttagaagttt cattgggtag 1740gaaacacatt tgaatggtat ttactaagat
actaaaatcc ttggacttca ctctaatttt 1800agtgccattt agaactcaag
gtctcagtaa aagtagaaat aaagcctgtt aacaaaacac 1860aagctgaata
ttaaaaatgt aactggattt tcaaagaaat gtttactggt attacctgta
1920gatgtatatt ctttattatg atcttttgtg taaagtctgg cagacaaatg
caatatctaa 1980ttgttgagtc caatatcaca agcagtacaa aagtataaaa
aagacttggc cttttctaat 2040gtgttaaaat actttatgct ggtaataaca
ctaagagtag ggcactagaa attttaagtg 2100aagataatgt gttgcagtta
ctgcactcaa tggcttacta ttataaacca aaactgggat 2160cactaagctc
cagtcagtca aaatgatcaa aattattgaa gagaataagc aattctgttc
2220tttattagga cacagtagat acagactaca aagtggagtg tgcttaataa
gaggtagcat 2280ttgttaagtg tcaattactc tattatccct tggagcttct
caaaataacc atataaggtg 2340taagatgtta aaggttatgg ttacactcag
tgcacaggta agctaatagg ctgagagaag 2400ctaaattact tactggggtc
tcacagtaag aaagtgagct gaagtttcag cccagattta 2460actggattct
gggctcttta ttcatgttac ttcatgaatc tgtttctcaa ttgtgcagaa
2520aaaagggggc tatttataag aaaagcaata aacaaacaag taatgatctc
aaataagtaa 2580tgcaagaaat agtgagattt caaaatcagt ggcagcgatt
tctcagttct gtcctaagtg 2640gccttgctca atcacctgct atcttttagt
ggagctttga aattatgttt cagacaactt 2700cgattcagtt ctagaatgtt
tgactcagca aattcacagg ctcatctttc taacttgatg 2760gtgaatatgg
aaattcagct aaatggatgt taataaaatt caaacgtttt aaggacagat
2820gaaaatgaca gaattttaag gtaaaatata tgaaggaata taagataaag
gatttttcta 2880ccttcagcaa aaacataccc actaattagt aaaattaata
ggcaaaaaaa agttgcatgc 2940tcttatactg taatgattat cattttaaaa
ctagcttttt gccttcgagc tatcggggta 3000aagacctaca ggaaaactac
tgtcgaaatc ctcgagggga agaaggggga ccctggtgtt 3060tcacaagcaa
tccagaggta cgctacgaag tctgtgacat tcctcagtgt tcagaagttg
3120aatgcatgac ctgcaatggg gagagttatc gaggtctcat ggatcataca
gaatcaggca 3180agatttgtca gcgctgggat catcagacac cacaccggca
caaattcttg cctgaaagat 3240atcccgacaa gggctttgat gataattatt
gccgcaatcc cgatggccag ccgaggccat 3300ggtgctatac tcttgaccct
cacacccgct gggagtactg tgcaattaaa acatgcgctg 3360acaatactat
gaatgacact gatgttcctt tggaaacaac tgaatgcatc caaggtcaag
3420gagaaggcta caggggcact gtcaatacca tttggaatgg aattccatgt
cagcgttggg 3480attctcagta tcctcacgag catgacatga ctcctgaaaa
tttcaagtgc aaggacctac 3540gagaaaatta ctgccgaaat ccagatgggt
ctgaatcacc ctggtgtttt accactgatc 3600caaacatccg agttggctac
tgctcccaaa ttccaaactg tgatatgtca catggacaag 3660attgttatcg
tgggaatggc aaaaattata tgggcaactt atcccaaaca agatctggac
3720taacatgttc aatgtgggac aagaacatgg aagacttaca tcgtcatatc
ttctgggaac 3780cagatgcaag taagctgaat gagaattact gccgaaatcc
agatgatgat gctcatggac 3840cctggtgcta cacgggaaat ccactcattc
cttgggatta ttgccctatt tctcgttgtg 3900aaggtgatac cacacctaca
atagtcaatt tagaccatcc cgtaatatct tgtgccaaaa 3960cgaaacaatt
gcgagttgta aatgggattc caacacgaac aaacatagga tggatggtta
4020gtttgagata cagaaataaa catatctgcg gaggatcatt gataaaggag
agttgggttc 4080ttactgcacg acagtgtttc ccttctcgag acttgaaaga
ttatgaagct tggcttggaa 4140ttcatgatgt ccacggaaga ggagatgaga
aatgcaaaca ggttctcaat gtttcccagc 4200tggtatatgg ccctgaagga
tcagatctgg ttttaatgaa gcttgccagg cctgctgtcc 4260tggatgattt
tgttagtacg attgatttac ctaattatgg atgcacaatt cctgaaaaga
4320ccagttgcag tgtttatggc tggggctaca ctggattgat caactatgat
ggcctattac 4380gagtggcaca tctctatata atgggaaatg agaaatgcag
ccagcatcat cgagggaagg 4440tgactctgaa tgagtctgaa atatgtgctg
gggctgaaaa gattggatca ggaccatgtg 4500agggggatta tggtggccca
cttgtttgtg agcaacataa aatgagaatg gttcttggtg 4560tcattgttcc
tggtcgtgga tgtgccattc caaatcgtcc tggtattttt gtccgagtag
4620catattatgc aaaatggata cacaaaatta ttttaacata taaggtacca
cagtcatag 467993679DNAArtificial SequenceSynthetic HGF-X7 gene
9atgtgggtga ccaaactcct gccagccctg ctgctgcagc atgtcctcct gcatctcctc
60ctgctcccca tcgccatccc ctatgcagag ggacaaagga aaagaagaaa tacaattcat
120gaattcaaaa aatcagcaaa gactacccta atcaaaatag atccagcact
gaagataaaa 180accaaaaaag tgaatactgc agaccaatgt gctaatagat
gtactaggaa taaaggactt 240ccattcactt gcaaggcttt tgtttttgat
aaagcaagaa aacaatgcct ctggttcccc 300ttcaatagca tgtcaagtgg
agtgaaaaaa gaatttggcc atgaatttga cctctatgaa 360aacaaagact
acattagaaa ctgcatcatc ggtaaaggac gcagctacaa gggaacagta
420tctatcacta agagtggcat caaatgtcag ccctggagtt ccatgatacc
acacgaacac 480aggtaagaac agtatgaaga aaagagatga agcctctgtc
ttttttacat gttaacagtc 540tcatattagt ccttcagaat aattctacaa
tcctaaaata acttagccaa cttgctgaat 600tgtattacgg caaggtttat
atgaattcat gactgatatt tagcaaatga ttaattaata 660tgttaataaa
atgtagccaa aacaatatct taccttaatg cctcaatttg tagatctcgg
720tatttgtgga tcctgggtag gaaacacatt tgaatggtat ttactaagat
actaaaatcc 780ttggacttca ctctaatttt agtgccattt agaactcaag
gtctcagtaa aagtagaaat 840aaagcctgtt aacaaaacac aagctgaata
ttaaaaatgt aactggattt tcaaagaaat 900gtttactggt attacctgta
gatgtatatt ctttattatg atcttttgtg taaagtctgg 960cagacaaatg
caatatctaa ttgttgagtc caatatcaca agcagtacaa aagtataaaa
1020aagacttggc cttttctaat gtgttaaaat actttatgct ggtaataaca
ctaagagtag 1080ggcactagaa
attttaagtg aagataatgt gttgcagtta ctgcactcaa tggcttacta
1140ttataaacca aaactgggat cactaagctc cagtcagtca aaatgatcaa
aattattgaa 1200gagaataagc aattctgttc tttattagga cacagtagat
acagactaca aagtggagtg 1260tgcttaataa gaggtagcat ttgttaagtg
tcaattactc tattatccct tggagcttct 1320caaaataacc atataaggtg
taagatgtta aaggttatgg ttacactcag tgcacaggta 1380agctaatagg
ctgagagaag ctaaattact tactggggtc tcacagtaag aaagtgagct
1440gaagtttcag cccagattta actggattct gggctcttta ttcatgttac
ttcatgaatc 1500tgtttctcaa ttgtgcagaa aaaagggggc tatttataag
aaaagcaata aacaaacaag 1560taatgatctc aaataagtaa tgcaagaaat
agtgagattt caaaatcagt ggcagcgatt 1620tctcagttct gtcctaagtg
gccttgctca atcacctgct atcttttagt ggagctttga 1680aattatgttt
cagacaactt cgattcagtt ctagaatgtt tgactcagca aattcacagg
1740ctcatctttc taacttgatg gtgaatatgg aaattcagct aaatggatgt
taataaaatt 1800caaacgtttt aaggacagat gaaaatgaca gaattttaag
gtaaaatata tgaaggaata 1860taagataaag gatttttcta ccttcagcaa
aaacataccc actaattagt aaaattaata 1920ggcaaaaaaa agttgcatgc
tcttatactg taatgattat cattttaaaa ctagcttttt 1980gccttcgagc
tatcggggta aagacctaca ggaaaactac tgtcgaaatc ctcgagggga
2040agaaggggga ccctggtgtt tcacaagcaa tccagaggta cgctacgaag
tctgtgacat 2100tcctcagtgt tcagaagttg aatgcatgac ctgcaatggg
gagagttatc gaggtctcat 2160ggatcataca gaatcaggca agatttgtca
gcgctgggat catcagacac cacaccggca 2220caaattcttg cctgaaagat
atcccgacaa gggctttgat gataattatt gccgcaatcc 2280cgatggccag
ccgaggccat ggtgctatac tcttgaccct cacacccgct gggagtactg
2340tgcaattaaa acatgcgctg acaatactat gaatgacact gatgttcctt
tggaaacaac 2400tgaatgcatc caaggtcaag gagaaggcta caggggcact
gtcaatacca tttggaatgg 2460aattccatgt cagcgttggg attctcagta
tcctcacgag catgacatga ctcctgaaaa 2520tttcaagtgc aaggacctac
gagaaaatta ctgccgaaat ccagatgggt ctgaatcacc 2580ctggtgtttt
accactgatc caaacatccg agttggctac tgctcccaaa ttccaaactg
2640tgatatgtca catggacaag attgttatcg tgggaatggc aaaaattata
tgggcaactt 2700atcccaaaca agatctggac taacatgttc aatgtgggac
aagaacatgg aagacttaca 2760tcgtcatatc ttctgggaac cagatgcaag
taagctgaat gagaattact gccgaaatcc 2820agatgatgat gctcatggac
cctggtgcta cacgggaaat ccactcattc cttgggatta 2880ttgccctatt
tctcgttgtg aaggtgatac cacacctaca atagtcaatt tagaccatcc
2940cgtaatatct tgtgccaaaa cgaaacaatt gcgagttgta aatgggattc
caacacgaac 3000aaacatagga tggatggtta gtttgagata cagaaataaa
catatctgcg gaggatcatt 3060gataaaggag agttgggttc ttactgcacg
acagtgtttc ccttctcgag acttgaaaga 3120ttatgaagct tggcttggaa
ttcatgatgt ccacggaaga ggagatgaga aatgcaaaca 3180ggttctcaat
gtttcccagc tggtatatgg ccctgaagga tcagatctgg ttttaatgaa
3240gcttgccagg cctgctgtcc tggatgattt tgttagtacg attgatttac
ctaattatgg 3300atgcacaatt cctgaaaaga ccagttgcag tgtttatggc
tggggctaca ctggattgat 3360caactatgat ggcctattac gagtggcaca
tctctatata atgggaaatg agaaatgcag 3420ccagcatcat cgagggaagg
tgactctgaa tgagtctgaa atatgtgctg gggctgaaaa 3480gattggatca
ggaccatgtg agggggatta tggtggccca cttgtttgtg agcaacataa
3540aatgagaatg gttcttggtg tcattgttcc tggtcgtgga tgtgccattc
caaatcgtcc 3600tggtattttt gtccgagtag catattatgc aaaatggata
cacaaaatta ttttaacata 3660taaggtacca cagtcatag
3679102729DNAArtificial SequenceSynthetic HGF-X8 gene 10atgtgggtga
ccaaactcct gccagccctg ctgctgcagc atgtcctcct gcatctcctc 60ctgctcccca
tcgccatccc ctatgcagag ggacaaagga aaagaagaaa tacaattcat
120gaattcaaaa aatcagcaaa gactacccta atcaaaatag atccagcact
gaagataaaa 180accaaaaaag tgaatactgc agaccaatgt gctaatagat
gtactaggaa taaaggactt 240ccattcactt gcaaggcttt tgtttttgat
aaagcaagaa aacaatgcct ctggttcccc 300ttcaatagca tgtcaagtgg
agtgaaaaaa gaatttggcc atgaatttga cctctatgaa 360aacaaagact
acattagaaa ctgcatcatc ggtaaaggac gcagctacaa gggaacagta
420tctatcacta agagtggcat caaatgtcag ccctggagtt ccatgatacc
acacgaacac 480aggtaagaac agtatgaaga aaagagatga agcctctgtc
ttttttacat gttaacagtc 540tcatattagt ccttcagaat aattctacaa
tcctaaaata acttagccaa cttgctgaat 600tgtattacgg caaggtttat
atgaattcat gactgatatt tagcaaatga ttaattaata 660tgttaataaa
atgtagccaa aacaatatct taccttaatg cctcaatttg tagatctcgg
720tatttgtgga tccttatgtt tcagacaact tcgattcagt tctagaatgt
ttgactcagc 780aaattcacag gctcatcttt ctaacttgat ggtgaatatg
gaaattcagc taaatggatg 840ttaataaaat tcaaacgttt taaggacaga
tgaaaatgac agaattttaa ggtaaaatat 900atgaaggaat ataagataaa
ggatttttct accttcagca aaaacatacc cactaattag 960taaaattaat
aggcaaaaaa aagttgcatg ctcttatact gtaatgatta tcattttaaa
1020actagctttt tgccttcgag ctatcggggt aaagacctac aggaaaacta
ctgtcgaaat 1080cctcgagggg aagaaggggg accctggtgt ttcacaagca
atccagaggt acgctacgaa 1140gtctgtgaca ttcctcagtg ttcagaagtt
gaatgcatga cctgcaatgg ggagagttat 1200cgaggtctca tggatcatac
agaatcaggc aagatttgtc agcgctggga tcatcagaca 1260ccacaccggc
acaaattctt gcctgaaaga tatcccgaca agggctttga tgataattat
1320tgccgcaatc ccgatggcca gccgaggcca tggtgctata ctcttgaccc
tcacacccgc 1380tgggagtact gtgcaattaa aacatgcgct gacaatacta
tgaatgacac tgatgttcct 1440ttggaaacaa ctgaatgcat ccaaggtcaa
ggagaaggct acaggggcac tgtcaatacc 1500atttggaatg gaattccatg
tcagcgttgg gattctcagt atcctcacga gcatgacatg 1560actcctgaaa
atttcaagtg caaggaccta cgagaaaatt actgccgaaa tccagatggt
1620ctgaatcacc ctggtgtttt accactgatc caaacatccg agttggctac
tgctcccaaa 1680ttccaaactg tgatatgtca catggacaag attgttatcg
tgggaatggc aaaaattata 1740tgggcaactt atcccaaaca agatctggac
taacatgttc aatgtgggac aagaacatgg 1800aagacttaca tcgtcatatc
ttctgggaac cagatgcaag taagctgaat gagaattact 1860gccgaaatcc
agatgatgat gctcatggac cctggtgcta cacgggaaat ccactcattc
1920cttgggatta ttgccctatt tctcgttgtg aaggtgatac cacacctaca
atagtcaatt 1980tagaccatcc cgtaatatct tgtgccaaaa cgaaacaatt
gcgagttgta aatgggattc 2040caacacgaac aaacatagga tggatggtta
gtttgagata cagaaataaa catatctgcg 2100gaggatcatt gataaaggag
agttgggttc ttactgcacg acagtgtttc ccttctcgag 2160acttgaaaga
ttatgaagct tggcttggaa ttcatgatgt ccacggaaga ggagatgaga
2220aatgcaaaca ggttctcaat gtttcccagc tggtatatgg ccctgaagga
tcagatctgg 2280ttttaatgaa gcttgccagg cctgctgtcc tggatgattt
tgttagtacg attgatttac 2340ctaattatgg atgcacaatt cctgaaaaga
ccagttgcag tgtttatggc tggggctaca 2400ctggattgat caactatgat
ggcctattac gagtggcaca tctctatata atgggaaatg 2460agaaatgcag
ccagcatcat cgagggaagg tgactctgaa tgagtctgaa atatgtgctg
2520gggctgaaaa gattggatca ggaccatgtg agggggatta tggtggccca
cttgtttgtg 2580agcaacataa aatgagaatg gttcttggtg tcattgttcc
tggtcgtgga tgtgccattc 2640caaatcgtcc tggtattttt gtccgagtag
catattatgc aaaatggata cacaaaatta 2700ttttaacata taaggtacca
cagtcatag 272911285PRTHomo sapiens 11Met Trp Val Thr Lys Leu Leu
Pro Ala Leu Leu Leu Gln His Val Leu1 5 10 15Leu His Leu Leu Leu Leu
Pro Ile Ala Ile Pro Tyr Ala Glu Gly Gln 20 25 30Arg Lys Arg Arg Asn
Thr Ile His Glu Phe Lys Lys Ser Ala Lys Thr 35 40 45Thr Leu Ile Lys
Ile Asp Pro Ala Leu Lys Ile Lys Thr Lys Lys Val 50 55 60Asn Thr Ala
Asp Gln Cys Ala Asn Arg Cys Thr Arg Asn Lys Gly Leu65 70 75 80Pro
Phe Thr Cys Lys Ala Phe Val Phe Asp Lys Ala Arg Lys Gln Cys 85 90
95Leu Trp Phe Pro Phe Asn Ser Met Ser Ser Gly Val Lys Lys Glu Phe
100 105 110 Gly His Glu Phe Asp Leu Tyr Glu Asn Lys Asp Tyr Ile Arg
Asn Cys 115 120 125Ile Ile Gly Lys Gly Arg Ser Tyr Lys Gly Thr Val
Ser Ile Thr Lys 130 135 140Ser Gly Ile Lys Cys Gln Pro Trp Ser Ser
Met Ile Pro His Glu His145 150 155 160Ser Tyr Arg Gly Lys Asp Leu
Gln Glu Asn Tyr Cys Arg Asn Pro Arg 165 170 175Gly Glu Glu Gly Gly
Pro Trp Cys Phe Thr Ser Asn Pro Glu Val Arg 180 185 190 Tyr Glu Val
Cys Asp Ile Pro Gln Cys Ser Glu Val Glu Cys Met Thr 195 200 205Cys
Asn Gly Glu Ser Tyr Arg Gly Leu Met Asp His Thr Glu Ser Gly 210 215
220Lys Ile Cys Gln Arg Trp Asp His Gln Thr Pro His Arg His Lys
Phe225 230 235 240Leu Pro Glu Arg Tyr Pro Asp Lys Gly Phe Asp Asp
Asn Tyr Cys Arg 245 250 255Asn Pro Asp Gly Gln Pro Arg Pro Trp Cys
Tyr Thr Leu Asp Pro His 260 265 270 Thr Arg Trp Glu Tyr Cys Ala Ile
Lys Thr Cys Glu Thr 275 280 28512296PRTHomo sapiens 12Met Trp Val
Thr Lys Leu Leu Pro Ala Leu Leu Leu Gln His Val Leu1 5 10 15Leu His
Leu Leu Leu Leu Pro Ile Ala Ile Pro Tyr Ala Glu Gly Gln 20 25 30Arg
Lys Arg Arg Asn Thr Ile His Glu Phe Lys Lys Ser Ala Lys Thr 35 40
45Thr Leu Ile Lys Ile Asp Pro Ala Leu Lys Ile Lys Thr Lys Lys Val
50 55 60Asn Thr Ala Asp Gln Cys Ala Asn Arg Cys Thr Arg Asn Lys Gly
Leu65 70 75 80Pro Phe Thr Cys Lys Ala Phe Val Phe Asp Lys Ala Arg
Lys Gln Cys 85 90 95Leu Trp Phe Pro Phe Asn Ser Met Ser Ser Gly Val
Lys Lys Glu Phe 100 105 110 Gly His Glu Phe Asp Leu Tyr Glu Asn Lys
Asp Tyr Ile Arg Asn Cys 115 120 125Ile Ile Gly Lys Gly Arg Ser Tyr
Lys Gly Thr Val Ser Ile Thr Lys 130 135 140Ser Gly Ile Lys Cys Gln
Pro Trp Ser Ser Met Ile Pro His Glu His145 150 155 160Ser Phe Leu
Pro Ser Ser Tyr Arg Gly Lys Asp Leu Gln Glu Asn Tyr 165 170 175Cys
Arg Asn Pro Arg Gly Glu Glu Gly Gly Pro Trp Cys Phe Thr Ser 180 185
190 Asn Pro Glu Val Arg Tyr Glu Val Cys Asp Ile Pro Gln Cys Ser Glu
195 200 205Val Glu Cys Met Thr Cys Asn Gly Glu Ser Tyr Arg Gly Leu
Met Asp 210 215 220His Thr Glu Ser Gly Lys Ile Cys Gln Arg Trp Asp
His Gln Thr Pro225 230 235 240His Arg His Lys Phe Leu Pro Glu Arg
Tyr Pro Asp Lys Gly Phe Asp 245 250 255Asp Asn Tyr Cys Arg Asn Pro
Asp Gly Gln Pro Arg Pro Trp Cys Tyr 260 265 270 Thr Leu Asp Pro His
Thr Arg Trp Glu Tyr Cys Ala Ile Lys Asn Met 275 280 285Arg Asp Ile
Thr Trp Ala Leu Asn 290 295136190DNAArtificial SequenceSynthetic
Hepatocyte Growth Factor Hybrid (HGF)-X2 13atgtgggtga ccaaactcct
gccagccctg ctgctgcagc atgtcctcct gcatctcctc 60ctgctcccca tcgccatccc
ctatgcagag ggacaaagga aaagaagaaa tacaattcat 120gaattcaaaa
aatcagcaaa gactacccta atcaaaatag atccagcact gaagataaaa
180accaaaaaag tgaatactgc agaccaatgt gctaatagat gtactaggaa
taaaggactt 240ccattcactt gcaaggcttt tgtttttgat aaagcaagaa
aacaatgcct ctggttcccc 300ttcaatagca tgtcaagtgg agtgaaaaaa
gaatttggcc atgaatttga cctctatgaa 360aacaaagact acattagaaa
ctgcatcatt ggtaaaggac gcagctacaa gggaacagta 420tctatcacta
agagtggcat caaatgtcag ccctggagtt ccatgatacc acacgaacac
480aggtaagaac agtatgaaga aaagagatga agcctctgtc ttttttacat
gttaacagtc 540tcatattagt ccttcagaat aattctacaa tcctaaaata
acttagccaa cttgctgaat 600tgtattacgg caaggtttat atgaattcat
gactgatatt tagcaaatga ttaattaata 660tgttaataaa atgtagccaa
aacaatatct taccttaatg cctcaatttg tagatctcgg 720tatttgtgaa
ataataacgt aaacttcgtt taaaaggatt cttcttcctg tctttgagaa
780agtacggcac tgtgcagggg gagaggttga ttgtgaaaaa tcagaggtag
atgagaatct 840tactgagggc tgagggttct ttaaccttgg tggatctcaa
cattggttgc acattaaaat 900cacctgctgc aagcccttga cgaatcttac
ttagaagatg acaacacaga acaattaaat 960cagaatctct ggggagaata
gggcaccagt attttttgag ctcccaccat gattccaaag 1020tgcagccaaa
tttgagaacc actgctaaaa gctcaagctt cagattgacc agcttttcca
1080tctcacctat cgcctaaaga ccaaattgga taaatgtgtt cattacgaca
gatgggtact 1140atttaaagat gagtaaacac aatatactta ggctcgtcag
actgagagtt ttaatcatca 1200ctgaggaaaa acatagatat ctaatactga
ctggagtatt agtcaaggct tatttcacac 1260acaattttat cagaaaccaa
agtagtttaa aacagctctc cccttattag taatgcattg 1320gagggtttac
tttaccatgt accttgctga gcactgtacc ttgttaatct catttacttg
1380taatgagaac cacacagcgg gtagttttat tggttctatt ttacctacat
gacaaaactg 1440aagcataaaa acacttagta agttttcagt gtcatgcaca
actaggaagt gacatggcca 1500gaatataagc ccagtcacca tcactctata
acctgcgctt ttaacaactt cagggcatga 1560cacatttggc cggtcagtag
aacccatgct gtgatttgtt tttgcagtgg tggtgatgac 1620tgccttgttg
aatccacttt ttattctatt ccattttggg gacacaattc tgcaagatga
1680ttcttcatta ggaaacagag atgagttatt gaccaacaca gaaagaaaaa
gagtttgttg 1740ctccacactg ggattaaacc tatgatcttg gcctaattaa
cactagctag taagtgtcca 1800agctgatcat ctctacaaca tttcaataac
agaaaacaac aattttcaaa attagttact 1860tacaattatg tagaaatgcc
tctaaaacac agtattttcc ttatattaca aaaacaaaaa 1920ttataattgg
ttttgtcctc ttttgagagt ttgcatggtg ttactccctg catagtgaag
1980aaaacatttt atttaagtag atggatctaa gtttttcatg aacaaaggaa
tgacatttga 2040aatcaatcct accctagtcc aggagaatgc attagattaa
cctagtagag gtcttatttc 2100accctgagtt ttctatgatc gtgattctct
gctggaggag taattgtgaa atagatctct 2160ctgggaactg gcttcctagt
ccaatcagct cttttaccaa tgaacacttc cttgtgatat 2220agatgtttat
ggccgagagg atcccttcct ttctacctgt atttgtccta ataaattgtt
2280gacttattaa ttcactactt cctcacagct tttttttggc tttacaaatc
cactggaaag 2340gtatatgggt gtatcacttt gtgtatttcg gtgtgcatgt
gtagagggga caaaaatcct 2400ctctcaaact ataaatattg agtatttgtg
tattgaacat ttgctataac tactaggttt 2460cttaaataat cttaatatat
aaaatgatat agaaaaaggg aaattatagt tcgtattatt 2520catctaagtg
aagagattaa aacccaggga gtaaataaat tgtctaagga ctaaggttgt
2580atactattta ggtgatagat atggggcaac cgtatgggtt ttatgattaa
caaataaact 2640tctcaccact ctaccatatc aacttttcca taaaagagag
ctatagtatt ctttgcttaa 2700ataaatttga ttagtgcatg acttcttgaa
aacatataaa gcaaaagtca catttgattc 2760tatcagaaaa gtgagtaagc
catggcccaa acaaaagatg cattaaaata ttctggaatg 2820atggagctaa
aagtaagaaa aatgactttt taaaaaagtt tactgttagg aattgtgaaa
2880ttatgctgaa ttttagttgc attataattt ttgtcagtca tacggtctga
caacctgtct 2940tatttctatt tccccatatg aggaatgcta gttaagtatg
gatattaact attactactt 3000agatgcattg aagttgcata atatggataa
tacttcactg gttccctgaa aatgtttagt 3060tagtaataag tctcttacac
tatttgtttt gtccaataat ttatattttc tgaagactta 3120actctagaat
acactcatgt caaaatgaaa gaatttcatt gcaaaatatt gcttggtaca
3180tgacgcatac ctgtatttgt tttgtgtcac aacatgaaaa atgatggttt
attagaagtt 3240tcattgggta ggaaacacat ttgaatggta tttactaaga
tactaaaatc cttggacttc 3300actctaattt tagtgccatt tagaactcaa
ggtctcagta aaagtagaaa taaagcctgt 3360taacaaaaca caaactgaat
attaaaaatg taactggatt ttcaaagaaa tgtttactgg 3420tattacctgt
agatgtatat tctttattat gatcttttgt gtaaagtctg gcagacaaat
3480gcaatatcta attgttgagt ccaatatcac aagcagtaca aaagtataaa
aaagacttgg 3540ccttttctaa tgtgttaaaa tactttatgc tggtaataac
actaagagta gggcactaga 3600aattttaagt gaagataatg tgttgcagtt
actgcactca atggcttact attataaacc 3660aaaactggga tcactaagct
ccagtcagtc aaaatgatca aaattattga agagaataag 3720caattctgtt
ctttattagg acacagtaga tacagactac aaagtggagt gtgcttaata
3780agaggtagca tttgttaagt gtcaattact ctattatccc ttggagcttc
tcaaaataac 3840catataaggt gtaagatgtt aaaggttatg gttacactca
gtgcacaggt aagctaatag 3900gctgagagaa gctaaattac ttactggggt
ctcacagtaa gaaagtgagc tgaagtttca 3960gcccagattt aactggattc
tgggctcttt attcatgtta cttcatgaat ctgtttctca 4020attgtgcaga
aaaaaggggg ctatttataa gaaaagcaat aaacaaacaa gtaatgatct
4080caaataagta atgcaagaaa tagtgagatt tcaaaatcag tggcagcgat
ttctcagttc 4140tgtcctaagt ggccttgctc aatcacctgc tatcttttag
tggagctttg aaattatgtt 4200tcagacaact tcgattcagt tctagaatgt
ttgactcagc aaattcacag gctcatcttt 4260ctaacttgat ggtgaatatg
gaaattcagc taaatggatg ttaataaaat tcaaacgttt 4320taaggacaga
tggaaatgac agaattttaa ggtaaaatat atgaaggaat ataagataaa
4380ggatttttct accttcagca aaaacatacc cactaattag taaaattaat
aggcgaaaaa 4440aagttgcatg ctcttatact gtaatgatta tcattttaaa
actagctttt tgccttcgag 4500ctatcggggt aaagacctac aggaaaacta
ctgtcgaaat cctcgagggg aagaaggggg 4560accctggtgt ttcacaagca
atccagaggt acgctacgaa gtctgtgaca ttcctcagtg 4620ttcagaagtt
gaatgcatga cctgcaatgg ggagagttat cgaggtctca tggatcatac
4680agaatcaggc aagatttgtc agcgctggga tcatcagaca ccacaccggc
acaaattctt 4740gcctgaaaga tatcccgaca agggctttga tgataattat
tgccgcaatc ccgatggcca 4800gccgaggcca tggtgctata ctcttgaccc
tcacacccgc tgggagtact gtgcaattaa 4860aacatgcgct gacaatacta
tgaatgacac tgatgttcct ttggaaacaa ctgaatgcat 4920ccaaggtcaa
ggagaaggct acaggggcac tgtcaatacc atttggaatg gaattccatg
4980tcagcgttgg gattctcagt atcctcacga gcatgacatg actcctgaaa
atttcaagtg 5040caaggaccta cgagaaaatt actgccgaaa tccagatggg
tctgaatcac cctggtgttt 5100taccactgat ccaaacatcc gagttggcta
ctgctcccaa attccaaact gtgatatgtc 5160acatggacaa gattgttatc
gtgggaatgg caaaaattat atgggcaact tatcccaaac 5220aagatctgga
ctaacatgtt caatgtggga caagaacatg gaagacttac atcgtcatat
5280cttctgggaa ccagatgcaa gtaagctgaa tgagaattac tgccgaaatc
cagatgatga 5340tgctcatgga ccctggtgct acacgggaaa tccactcatt
ccttgggatt attgccctat 5400ttctcgttgt gaaggtgata ccacacctac
aatagtcaat ttagaccatc ccgtaatatc 5460ttgtgccaaa acgaaacaat
tgcgagttgt aaatgggatt ccaacacgaa caaacatagg 5520atggatggtt
agtttgagat acagaaataa acatatctgc ggaggatcat tgataaagga
5580gagttgggtt cttactgcac gacagtgttt cccttctcga gacttgaaag
attatgaagc 5640ttggcttgga attcatgatg tccacggaag aggagatgag
aaatgcaaac aggttctcaa 5700tgtttcccag ctggtatatg gccctgaagg
atcagatctg gttttaatga agcttgccag 5760gcctgctgtc ctggatgatt
ttgttagtac gattgattta cctaattatg gatgcacaat 5820tcctgaaaag
accagttgca gtgtttatgg ctggggctac actggattga tcaactatga
5880tggcctatta cgagtggcac atctctatat aatgggaaat gagaaatgca
gccagcatca
5940tcgagggaag gtgactctga atgagtctga aatatgtgct ggggctgaaa
agattggatc 6000aggaccatgt gagggggatt atggtggccc acttgtttgt
gagcaacata aaatgagaat 6060ggttcttggt gtcattgttc ctggtcgtgg
atgtgccatt ccaaatcgtc ctggtatttt 6120tgtccgagta gcatattatg
caaaatggat acacaaaatt attttaacat ataaggtacc 6180acagtcatag
6190145190DNAArtificial SequenceSynthetic Hepatocyte Growth Factor
Hybrid (HGF)-X3 14atgtgggtga ccaaactcct gccagccctg ctgctgcagc
atgtcctcct gcatctcctc 60ctgctcccca tcgccatccc ctatgcagag ggacaaagga
aaagaagaaa tacaattcat 120gaattcaaaa aatcagcaaa gactacccta
atcaaaatag atccagcact gaagataaaa 180accaaaaaag tgaatactgc
agaccaatgt gctaatagat gtactaggaa taaaggactt 240ccattcactt
gcaaggcttt tgtttttgat aaagcaagaa aacaatgcct ctggttcccc
300ttcaatagca tgtcaagtgg agtgaaaaaa gaatttggcc atgaatttga
cctctatgaa 360aacaaagact acattagaaa ctgcatcatt ggtaaaggac
gcagctacaa gggaacagta 420tctatcacta agagtggcat caaatgtcag
ccctggagtt ccatgatacc acacgaacac 480aggtaagaac agtatgaaga
aaagagatga agcctctgtc ttttttacat gttaacagtc 540tcatattagt
ccttcagaat aattctacaa tcctaaaata acttagccaa cttgctgaat
600tgtattacgg caaggtttat atgaattcat gactgatatt tagcaaatga
ttaattaata 660tgttaataaa atgtagccaa aacaatatct taccttaatg
cctcaatttg tagatctcgg 720tatttgtgaa ataataacgt aaacttcgtt
taaaaggatt cttcttcctg tctttgagaa 780agtacggcac tgtgcagggg
gagaggttga ttgtgaaaaa tcagaggtag atgagaatct 840tactgagggc
tgagggttct ttaaccttgg tggatctcaa cattggttgc acattaaaat
900cacctgctgc aagcccttga cgaatcttac ttagaagatg acaacacaga
acaattaaat 960cagaatctct ggggagaata gggcaccagt attttttgag
ctcccaccat gattccaaag 1020tgcagccaaa tttgagaacc actgctaaaa
gctcaagctt cagattgacc agcttttcca 1080tctcacctat cgcctaaaga
ccaaattgga taaatgtgtt cattacgaca gatgggtact 1140atttaaagat
gagtaaacac aatatactta ggctcgtcag actgagagtt ttaatcatca
1200ctgaggaaaa acatagatat ctaatactga ctggagtatt agtcaaggct
tatttcacac 1260acaattttat cagaaaccaa agtagtttaa aacagctctc
cccttattag taatgcattg 1320gagggtttac tttaccatgt accttgctga
gcactgtacc ttgttaatct catttacttg 1380taatgagaac cacacagcgg
gtagttttat tggttctatt ttacctacat gacaaaactg 1440aagcataaaa
acacttagta agttttcagt gtcatgcaca actaggaagt gacatggcca
1500gaatataagc ccagtcacca tcactctata acctgcgctt ttaacaactt
cagggcatga 1560cacatttggc cggtcagtag aacccatgct gtgatttgtt
tttgcagtgg tggtgatgac 1620tgccttgttg aatccacttt ttattctatt
ccattttggg gacacaattc tgcaagatga 1680ttcttcatta ggaaacagag
atgagttatt gaccaacaca gaaagaaaaa gagtttgttg 1740ctccacactg
ggattaaacc tatgatcttg gcctaattaa cactagctag taagtgtcca
1800agctgatcat ctctacaaca tttcaataac agaaaacaac aattttcaaa
attagttact 1860tacaattatg tagaaatgcc tctaaaacac agtattttcc
ttatattaca aaaacaaaaa 1920ttataattgg ttttgtcctc ttttgagagt
ttgcatggtg ttactccctg catagtgaag 1980aaaacatttt atttaagtag
atggatctaa gtttttcatg aacaaaggaa tgacatttga 2040aatcaatcct
accctagtcc aggagaatgc attagattaa cctagtagag gtcttatttc
2100accctgagtt ttctatgatc gtgattctct gctggaggag taattgtgaa
atagatctct 2160ctgggaactg gcttcctagt ccaatcagct cttttaccaa
tgaacacttc cttgtgatat 2220agatgtttat ggccgagagg atcctgggta
ggaaacacat ttgaatggta tttactaaga 2280tactaaaatc cttggacttc
actctaattt tagtgccatt tagaactcaa ggtctcagta 2340aaagtagaaa
taaagcctgt taacaaaaca caaactgaat attaaaaatg taactggatt
2400ttcaaagaaa tgtttactgg tattacctgt agatgtatat tctttattat
gatcttttgt 2460gtaaagtctg gcagacaaat gcaatatcta attgttgagt
ccaatatcac aagcagtaca 2520aaagtataaa aaagacttgg ccttttctaa
tgtgttaaaa tactttatgc tggtaataac 2580actaagagta gggcactaga
aattttaagt gaagataatg tgttgcagtt actgcactca 2640atggcttact
attataaacc aaaactggga tcactaagct ccagtcagtc aaaatgatca
2700aaattattga agagaataag caattctgtt ctttattagg acacagtaga
tacagactac 2760aaagtggagt gtgcttaata agaggtagca tttgttaagt
gtcaattact ctattatccc 2820ttggagcttc tcaaaataac catataaggt
gtaagatgtt aaaggttatg gttacactca 2880gtgcacaggt aagctaatag
gctgagagaa gctaaattac ttactggggt ctcacagtaa 2940gaaagtgagc
tgaagtttca gcccagattt aactggattc tgggctcttt attcatgtta
3000cttcatgaat ctgtttctca attgtgcaga aaaaaggggg ctatttataa
gaaaagcaat 3060aaacaaacaa gtaatgatct caaataagta atgcaagaaa
tagtgagatt tcaaaatcag 3120tggcagcgat ttctcagttc tgtcctaagt
ggccttgctc aatcacctgc tatcttttag 3180tggagctttg aaattatgtt
tcagacaact tcgattcagt tctagaatgt ttgactcagc 3240aaattcacag
gctcatcttt ctaacttgat ggtgaatatg gaaattcagc taaatggatg
3300ttaataaaat tcaaacgttt taaggacaga tggaaatgac agaattttaa
ggtaaaatat 3360atgaaggaat ataagataaa ggatttttct accttcagca
aaaacatacc cactaattag 3420taaaattaat aggcgaaaaa aagttgcatg
ctcttatact gtaatgatta tcattttaaa 3480actagctttt tgccttcgag
ctatcggggt aaagacctac aggaaaacta ctgtcgaaat 3540cctcgagggg
aagaaggggg accctggtgt ttcacaagca atccagaggt acgctacgaa
3600gtctgtgaca ttcctcagtg ttcagaagtt gaatgcatga cctgcaatgg
ggagagttat 3660cgaggtctca tggatcatac agaatcaggc aagatttgtc
agcgctggga tcatcagaca 3720ccacaccggc acaaattctt gcctgaaaga
tatcccgaca agggctttga tgataattat 3780tgccgcaatc ccgatggcca
gccgaggcca tggtgctata ctcttgaccc tcacacccgc 3840tgggagtact
gtgcaattaa aacatgcgct gacaatacta tgaatgacac tgatgttcct
3900ttggaaacaa ctgaatgcat ccaaggtcaa ggagaaggct acaggggcac
tgtcaatacc 3960atttggaatg gaattccatg tcagcgttgg gattctcagt
atcctcacga gcatgacatg 4020actcctgaaa atttcaagtg caaggaccta
cgagaaaatt actgccgaaa tccagatggg 4080tctgaatcac cctggtgttt
taccactgat ccaaacatcc gagttggcta ctgctcccaa 4140attccaaact
gtgatatgtc acatggacaa gattgttatc gtgggaatgg caaaaattat
4200atgggcaact tatcccaaac aagatctgga ctaacatgtt caatgtggga
caagaacatg 4260gaagacttac atcgtcatat cttctgggaa ccagatgcaa
gtaagctgaa tgagaattac 4320tgccgaaatc cagatgatga tgctcatgga
ccctggtgct acacgggaaa tccactcatt 4380ccttgggatt attgccctat
ttctcgttgt gaaggtgata ccacacctac aatagtcaat 4440ttagaccatc
ccgtaatatc ttgtgccaaa acgaaacaat tgcgagttgt aaatgggatt
4500ccaacacgaa caaacatagg atggatggtt agtttgagat acagaaataa
acatatctgc 4560ggaggatcat tgataaagga gagttgggtt cttactgcac
gacagtgttt cccttctcga 4620gacttgaaag attatgaagc ttggcttgga
attcatgatg tccacggaag aggagatgag 4680aaatgcaaac aggttctcaa
tgtttcccag ctggtatatg gccctgaagg atcagatctg 4740gttttaatga
agcttgccag gcctgctgtc ctggatgatt ttgttagtac gattgattta
4800cctaattatg gatgcacaat tcctgaaaag accagttgca gtgtttatgg
ctggggctac 4860actggattga tcaactatga tggcctatta cgagtggcac
atctctatat aatgggaaat 4920gagaaatgca gccagcatca tcgagggaag
gtgactctga atgagtctga aatatgtgct 4980ggggctgaaa agattggatc
aggaccatgt gagggggatt atggtggccc acttgtttgt 5040gagcaacata
aaatgagaat ggttcttggt gtcattgttc ctggtcgtgg atgtgccatt
5100ccaaatcgtc ctggtatttt tgtccgagta gcatattatg caaaatggat
acacaaaatt 5160attttaacat ataaggtacc acagtcatag 5190
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