U.S. patent application number 13/638490 was filed with the patent office on 2013-05-23 for cancer treatment with recombinant vector.
This patent application is currently assigned to TOCAGEN INC.. The applicant listed for this patent is Harry E. Gruber, Douglas J. Jolly, Derek G. Ostertag, Omar Perez, Joan M. Robbins. Invention is credited to Harry E. Gruber, Douglas J. Jolly, Derek G. Ostertag, Omar Perez, Joan M. Robbins.
Application Number | 20130130986 13/638490 |
Document ID | / |
Family ID | 44763495 |
Filed Date | 2013-05-23 |
United States Patent
Application |
20130130986 |
Kind Code |
A1 |
Gruber; Harry E. ; et
al. |
May 23, 2013 |
CANCER TREATMENT WITH RECOMBINANT VECTOR
Abstract
This disclosure provides modified cytosine deaminases(CDs). The
disclosure further relates to cells and vector expressing or
comprising such modified CDs and methods of using such modified CDs
in the treatment of disease and disorders. It further provides use
of such modified CDs with a thymosin-alpha-1 polypeptide in the
treatment of disease and disorders.
Inventors: |
Gruber; Harry E.; (Rancho
Santa Fe, CA) ; Jolly; Douglas J.; (Encinitas,
CA) ; Perez; Omar; (San Diego, CA) ; Ostertag;
Derek G.; (San Diego, CA) ; Robbins; Joan M.;
(San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Gruber; Harry E.
Jolly; Douglas J.
Perez; Omar
Ostertag; Derek G.
Robbins; Joan M. |
Rancho Santa Fe
Encinitas
San Diego
San Diego
San Diego |
CA
CA
CA
CA
CA |
US
US
US
US
US |
|
|
Assignee: |
TOCAGEN INC.
San Diego
CA
|
Family ID: |
44763495 |
Appl. No.: |
13/638490 |
Filed: |
March 29, 2011 |
PCT Filed: |
March 29, 2011 |
PCT NO: |
PCT/US11/30402 |
371 Date: |
January 16, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61318728 |
Mar 29, 2010 |
|
|
|
Current U.S.
Class: |
514/12.9 |
Current CPC
Class: |
A61K 48/00 20130101;
C12N 2760/16032 20130101; A61K 31/513 20130101; A61P 35/00
20180101; C12N 7/00 20130101; C12N 9/78 20130101; C12N 2760/16021
20130101; A61K 38/2292 20130101; C12N 2760/16043 20130101; C12N
15/86 20130101; C12N 2740/13043 20130101 |
Class at
Publication: |
514/12.9 |
International
Class: |
A61K 48/00 20060101
A61K048/00; A61K 38/22 20060101 A61K038/22 |
Claims
1. A therapeutic combination comprising a thymosin-1-alpha
polypeptide and a replication retroviral vector for use in the
treatment of a subject comprising a cell proliferative disease or
disorder, wherein the replication competent retroviral vector
comprises a retroviral GAG protein; a retroviral POL protein; a
retroviral envelope; a retroviral polynucleotide comprising
Long-Terminal Repeat (LTR) sequences at the 3' end of the
retroviral polynucleotide sequence, a promoter sequence at the 5'
end of the retroviral polynucleotide, said promoter being suitable
for expression in a mammalian cell, a gag nucleic acid domain, a
pol nucleic acid domain and an env nucleic acid domain; a cassette
comprising an internal ribosome entry site (IRES) operably linked
to a heterologous polynucleotide, wherein the cassette is
positioned 5' to the 3' LTR and 3' to the env nucleic acid domain
encoding the retroviral envelope; and cis-acting sequences
necessary for reverse transcription, packaging and integration in a
target cell.
2. The combination of claim 1, wherein the heterologous
polynucleotide comprises a suicide gene that expresses a
polypeptide that converts a non-toxic prodrug to a toxic drug.
3. The combination of claim 1, wherein the target cell is a cancer
cell.
4. The combination of claim 1, wherein the target cell comprises a
cell proliferative disorder.
5. The combination of claim 4, wherein the cell proliferative
disorder is selected from the group consisting of lung cancer,
colon-rectum cancer, breast cancer, prostate cancer, urinary tract
cancer, uterine cancer, brain cancer, head and neck cancer,
pancreatic cancer, melanoma, stomach cancer and ovarian cancer,
rheumatoid arthritis or other autoimmune disease.
6. The combination of claim 1, wherein the retroviral vector is
administered prior to the thymosin-alpha-1 polypeptide.
7. The combination of claim 1, wherein the retroviral
polynucleotide sequence is derived from murine leukemia virus
(MLV), Moloney murine leukemia virus (MoMLV), Feline leukemia virus
(FeLV) Baboon endogenous retrovirus (BEV), porcine endogenous virus
(PERV), the cat derived retrovirus RD114, squirrel monkey
retrovirus, Xenotropic murine leukemia virus-related virus (XMRV),
avian reticuloendotheliosis virus (REV), or Gibbon ape leukemia
virus (GALV).
8. The combination of claim 1, wherein the retroviral envelope is
an amphotropic MLV envelope.
9. The combination of claim 1, wherein the retrovirus is a
gammaretrovirus.
10. The combination of claim 1, wherein the thymosin-alpha-1
polypeptide comprises at least 85% identity to SEQ ID NO:73 and
having thymosin-alpha-1 activity.
11. The combination of claim 1, wherein the heterologous
polynucleotide encodes a polypeptide having cytosine deaminase
activity.
12. The combination of claim 1, wherein the heterologous
polynucleotide is selected from the group consisting of a suicide
gene and an immunopotentiating gene.
13. The combination of claim 1, wherein the retrovirus further
comprises an miRNA.
14. The combination of claim 1, wherein the replication competent
retrovirus comprises a retroviral GAG protein; a retroviral POL
protein; a retroviral envelope; a retroviral polynucleotide
comprising Long-Terminal Repeat (LTR) sequences at the 3' end of
the retroviral polynucleotide sequence, a promoter sequence at the
5' end of the retroviral polynucleotide, said promoter being
suitable for expression in a mammalian cell, a gag nucleic acid
domain, a pol nucleic acid domain and an env nucleic acid domain; a
cassette comprising an internal ribosome entry site (IRES) operably
linked to a polynucleotide encoding cytosine deaminase, wherein the
cassette is positioned 5' to the 3' LTR and 3' to the env nucleic
acid domain encoding the retroviral envelope; and cis-acting
sequences necessary for reverse transcription, packaging and
integration in a target cell.
15. The combination of claim 1, wherein the thymosin-1-alpha and
retroviral vector are formulated for delivery simultaneously.
16. A method of treating a subject with a cell proliferative
disorder comprising: administering a thymosin-alpha-1 polypeptide
to the subject either before, during or after administration of a
replication competent retrovirus, the replication competent
retrovirus comprising a retroviral GAG protein; a retroviral POL
protein; a retroviral envelope; a retroviral polynucleotide
comprising Long-Terminal Repeat (LTR) sequences at the 3' end of
the retroviral polynucleotide sequence, a promoter sequence at the
5' end of the retroviral polynucleotide, said promoter being
suitable for expression in a mammalian cell, a gag nucleic acid
domain, a pol nucleic acid domain and an env nucleic acid domain; a
cassette comprising an internal ribosome entry site (IRES) operably
linked to a heterologous polynucleotide, wherein the cassette is
positioned 5' to the 3' LTR and 3' to the env nucleic acid domain
encoding the retroviral envelope; and cis-acting sequences
necessary for reverse transcription, packaging and integration in a
target cell.
17. The method of claim 16, wherein the retroviral polynucleotide
sequence is derived from murine leukemia virus (MLV), Moloney
murine leukemia virus (MoMLV), Feline leukemia virus (FeLV) Baboon
endogenous retrovirus (BEV), porcine endogenous virus (PERV), the
cat derived retrovirus RD114, squirrel monkey retrovirus,
Xenotropic murine leukemia virus-related virus (XMRV), avian
reticuloendotheliosis virus (REV), or Gibbon ape leukemia virus
(GALV).
18. The method of claim 16, wherein the retroviral envelope is an
amphotropic MLV envelope.
19. The method of claim 16, wherein the retrovirus is a
gammaretrovirus.
20. The method of claim 16, wherein the target cell is a neoplastic
cell.
21. The method of claim 16, wherein the cell proliferative disorder
is selected from the group consisting of lung cancer, colon-rectum
cancer, breast cancer, prostate cancer, urinary tract cancer,
uterine cancer, brain cancer, head and neck cancer, pancreatic
cancer, melanoma, stomach cancer and ovarian cancer, rheumatoid
arthritis or other autoimmune disease.
22. The method of claim 16, wherein the thymosin-alpha-1
polypeptide comprises at least 85% identity to SEQ ID NO:73 and
having a thymosin-alpha-1 activity.
23. The method of claim 16, wherein the heterologous polynucleotide
encodes a polypeptide having cytosine deaminase activity.
24. The method of claim 16, wherein the heterologous polynucleotide
is selected from the group consisting of a suicide gene and an
immunopotentiating gene.
25. The method of claim 16, wherein the retrovirus further
comprises an miRNA.
26. The method of claim 16, wherein the replication competent
retrovirus comprising a retroviral GAG protein; a retroviral POL
protein; a retroviral envelope; a retroviral polynucleotide
comprising Long-Terminal Repeat (LTR) sequences at the 3' end of
the retroviral polynucleotide sequence, a promoter sequence at the
5' end of the retroviral polynucleotide, said promoter being
suitable for expression in a mammalian cell, a gag nucleic acid
domain, a pol nucleic acid domain and an env nucleic acid domain; a
cassette comprising an internal ribosome entry site (IRES) operably
linked to a polynucleotide encoding cytosine deaminase, wherein the
cassette is positioned 5' to the 3' LTR and 3' to the env nucleic
acid domain encoding the retroviral envelope; and cis-acting
sequences necessary for reverse transcription, packaging and
integration in a target cell.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application Ser. No. 61/318,728, filed Mar. 29, 2010, the
disclosure of which is incorporated herein by reference.
TECHNICAL FIELD
[0002] This disclosure relates to replication competent retroviral
vectors for treating cell proliferative. The disclosure further
relates to the use of such replication competent retroviral vectors
and factors for delivery and expression of heterologous nucleic
acids.
BACKGROUND
[0003] Effective methods of delivering genes and heterologous
nucleic acids to cells and subjects has been a goal researchers for
scientific development and for possible treatments of diseases and
disorders.
SUMMARY
[0004] The disclosure provides a therapeutic combination comprising
a thymosin-1-alpha polypeptide and a replication retroviral vector
for use in the treatment of a subject comprising a cell
proliferative disease or disorder, wherein the replication
competent retroviral vector comprises a retroviral GAG protein; a
retroviral POL protein; a retroviral envelope; a retroviral
polynucleotide comprising Long-Terminal Repeat (LTR) sequences at
the 3' end of the retroviral polynucleotide sequence, a promoter
sequence at the 5' end of the retroviral polynucleotide, said
promoter being suitable for expression in a mammalian cell, a gag
nucleic acid domain, a pol nucleic acid domain and an env nucleic
acid domain; a cassette comprising an internal ribosome entry site
(IRES) operably linked to a heterologous polynucleotide, wherein
the cassette is positioned 5' to the 3' LTR and 3' to the env
nucleic acid domain encoding the retroviral envelope; and
cis-acting sequences necessary for reverse transcription, packaging
and integration in a target cell. In one embodiment, the
heterologous polynucleotide comprises a suicide gene that expresses
a polypeptide that converts a non-toxic prodrug to a toxic drug. In
another embodiment, the target cell is a cancer cell. In yet
another embodiment, the target cell comprises a cell proliferative
disorder. In a further embodiment, the cell proliferative disorder
is selected from the group consisting of lung cancer, colon-rectum
cancer, breast cancer, prostate cancer, urinary tract cancer,
uterine cancer, brain cancer, head and neck cancer, pancreatic
cancer, melanoma, stomach cancer and ovarian cancer, rheumatoid
arthritis or other autoimmune disease. In one embodiment, the
retroviral vector is administered prior to the thymosin-alpha-1
polypeptide. In another embodiment, the retroviral polynucleotide
sequence is derived from murine leukemia virus (MLV), Moloney
murine leukemia virus (MoMLV), Feline leukemia virus (FeLV) Baboon
endogenous retrovirus (BEV), porcine endogenous virus (PERV), the
cat derived retrovirus RD114, squirrel monkey retrovirus,
Xenotropic murine leukemia virus-related virus (XMRV), avian
reticuloendotheliosis virus (REV), or Gibbon ape leukemia virus
(GALV). In yet another embodiment, the retroviral envelope is an
amphotropic MLV envelope. In one embodiment, the retrovirus is a
gammaretrovirus. In another embodiment, the thymosin-alpha-1
polypeptide comprises at least 85% identity to SEQ ID NO:73 and
having a thymosin-alpha-1 activity. In yet another embodiment, the
heterologous polynucleotide encodes a polypeptide having cytosine
deaminase activity. In one embodiment, the heterologous
polynucleotide is selected from the group consisting of a suicide
gene and an immunopotentiating gene. In any of the foregoing
embodiments, the retrovirus further comprises an miRNA. In a
specific embodiment, the replication competent retrovirus
comprising a retroviral GAG protein; a retroviral POL protein; a
retroviral envelope; a retroviral polynucleotide comprising
Long-Terminal Repeat (LTR) sequences at the 3' end of the
retroviral polynucleotide sequence, a promoter sequence at the 5'
end of the retroviral polynucleotide, said promoter being suitable
for expression in a mammalian cell, a gag nucleic acid domain, a
pol nucleic acid domain and an env nucleic acid domain; a cassette
comprising an internal ribosome entry site (IRES) operably linked
to a polynucleotide encoding cytosine deaminase, wherein the
cassette is positioned 5' to the 3' LTR and 3' to the env nucleic
acid domain encoding the retroviral envelope; and cis-acting
sequences necessary for reverse transcription, packaging and
integration in a target cell. In any of the foregoing embodiments,
the thymosin-1-alpha and retroviral vector are formulated for
delivery simultaneously.
[0005] The disclosure also provides a method of treating a subject
with a cell proliferative disorder comprising administering a
thymosin-alpha-1 polypeptide to the subject either before, during
or after administration of a replication competent retrovirus
comprising a retroviral GAG protein; a retroviral POL protein; a
retroviral envelope; a retroviral polynucleotide comprising
Long-Terminal Repeat (LTR) sequences at the 3' end of the
retroviral polynucleotide sequence, a promoter sequence at the 5'
end of the retroviral polynucleotide, said promoter being suitable
for expression in a mammalian cell, a gag nucleic acid domain, a
pol nucleic acid domain and an env nucleic acid domain; a cassette
comprising an internal ribosome entry site (IRES) operably linked
to a heterologous polynucleotide, wherein the cassette is
positioned 5' to the 3' LTR and 3' to the env nucleic acid domain
encoding the retroviral envelope; and cis-acting sequences
necessary for reverse transcription, packaging and integration in a
target cell. In one embodiment, the heterologous polynucleotide
comprises a suicide gene that expresses a polypeptide that converts
a non-toxic prodrug to a toxic drug. In another embodiment, the
target cell is a cancer cell. In yet another embodiment, the target
cell comprises a cell proliferative disorder. In a further
embodiment, the cell proliferative disorder is selected from the
group consisting of lung cancer, colon-rectum cancer, breast
cancer, prostate cancer, urinary tract cancer, uterine cancer,
brain cancer, head and neck cancer, pancreatic cancer, melanoma,
stomach cancer and ovarian cancer, rheumatoid arthritis or other
autoimmune disease. In one embodiment, the retroviral vector is
administered prior to the thymosin-alpha-1 polypeptide. In another
embodiment, the retroviral polynucleotide sequence is derived from
murine leukemia virus (MLV), Moloney murine leukemia virus (MoMLV),
Feline leukemia virus (FeLV) Baboon endogenous retrovirus (BEV),
porcine endogenous virus (PERV), the cat derived retrovirus RD114,
squirrel monkey retrovirus, Xenotropic murine leukemia
virus-related virus (XMRV), avian reticuloendotheliosis virus
(REV), or Gibbon ape leukemia virus (GALV). In yet another
embodiment, the retroviral envelope is an amphotropic MLV envelope.
In one embodiment, the retrovirus is a gammaretrovirus. In another
embodiment, the thymosin-alpha-1 polypeptide comprises at least 85%
identity to SEQ ID NO:73 and having a thymosin-alpha-1 activity. In
yet another embodiment, the heterologous polynucleotide encodes a
polypeptide having cytosine deaminase activity. In one embodiment,
the heterologous polynucleotide is selected from the group
consisting of a suicide gene and an immunopotentiating gene. In any
of the foregoing embodiments, the retrovirus further comprises an
miRNA. In a specific embodiment, the replication competent
retrovirus comprising a retroviral GAG protein; a retroviral POL
protein; a retroviral envelope; a retroviral polynucleotide
comprising Long-Terminal Repeat (LTR) sequences at the 3' end of
the retroviral polynucleotide sequence, a promoter sequence at the
5' end of the retroviral polynucleotide, said promoter being
suitable for expression in a mammalian cell, a gag nucleic acid
domain, a pol nucleic acid domain and an env nucleic acid domain; a
cassette comprising an internal ribosome entry site (IRES) operably
linked to a polynucleotide encoding cytosine deaminase, wherein the
cassette is positioned 5' to the 3' LTR and 3' to the env nucleic
acid domain encoding the retroviral envelope; and cis-acting
sequences necessary for reverse transcription, packaging and
integration in a target cell. In any of the foregoing embodiments,
the thymosin-1-alpha and retroviral vector are formulated for
delivery simultaneously.
[0006] The details of one or more embodiments of the disclosure are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages will be apparent from the
description and drawings, and from the claims.
BRIEF DESCRIPTION OF DRAWINGS
[0007] FIG. 1A-C shows an alignment of the Wild-type yeast cytosine
deaminase (SEQ ID NO: 2) and a cytosine deaminase of the disclosure
(SEQ ID NO: 4) and other sequences of the disclosure (SEQ ID
NOs:31-40).
[0008] FIG. 2 shows a graph of cell killing data showing that
modified vectors are more effective compared to the original wild
type CD. The graph also shows that the new modified backbone
(T5.0007) is more effective at killing than the old backbone
(pACE-CD). Also shown is a table cataloguing the various vector
constructs and their names
[0009] FIG. 3A-F shows (A) a schematic of a recombinant retroviral
vector of the disclosure; (B) and (C) are plasmid maps of a
polynucleotide of the disclosure. (D) sequence of the vector
encoding part of pAC3-yCD2 of the disclosure. (E) schematics of
recombinant vectors of the disclosure and (F) plasmid maps of
vectors of the disclosure.
[0010] FIG. 4 shows that higher levels of yCD2 protein are observed
compared to wild type yCD protein in infected U-87 cells.
[0011] FIG. 5 shows that a vector of the disclosure is genetically
stable after 12 cycles of viral passages as assessed using PCR
amplification. The figure also demonstrates that the vectors of the
disclosure are more stable after longer passages compared to the
vector pACE-CD (Kasahara et al.). In particular pAC3-CD is more
stable than pACE-CD, demonstrating that the changed backbone has
made the vector more stable. In addition pACE-yCD1 (T5.0001) and
-yCD2 (T5-0002) are very much more stable than pAC-yCD,
demonstrating that small and silent changes to the coding sequence
of the transgene can have a very large effect on stability, leading
to superior properties.
[0012] FIG. 6A-B shows cell killing assays and cytosine deaminase
specific activity of cells infected with different vectors. (A)
shows that cytosine deaminase and vector of the disclosure kill
infected cells at least as well and perhaps better than the
original pACE-CD when U87 infected cells are exposed to increasing
levels of 5-FC. (B) shows that the specific CD activity of the
disclosure (T5.0007, T5.0001 and T5.0002) are all increased
compared to pACE-CD (T5.0000), and is in the order
T5.0000<T5.0007<T5.0001<T5.0002.
[0013] FIG. 7 shows U-87 tumors treated with CD vector of the
disclosure in vivo and explanted from mice treated with 4 cycles of
5-FC are still sensitive to the drug.
[0014] FIG. 8 shows dosing information and therapeutic effect in a
Kaplan-Meyer survival analysis in a mouse model of brain
cancer.
[0015] FIG. 9 shows dosing information and therapeutic effect in a
Kaplan-Meyer survival analysis in a syngeneic mouse model.
[0016] FIG. 10 shows a survival curve for combination therapy with
thymosin alpha 1 and a replication competent retrovirus of the
disclosure expressing cytosine deaminase.
DETAILED DESCRIPTION
[0017] As used herein and in the appended claims, the singular
forms "a," "and," and "the" include plural referents unless the
context clearly dictates otherwise. Thus, for example, reference to
"a cell" includes a plurality of such cells and reference to "the
agent" includes reference to one or more agents known to those
skilled in the art, and so forth.
[0018] Also, the use of "or" means "and/or" unless stated
otherwise. Similarly, "comprise," "comprises," "comprising"
"include," "includes," and "including" are interchangeable and not
intended to be limiting.
[0019] It is to be further understood that where descriptions of
various embodiments use the term "comprising," those skilled in the
art would understand that in some specific instances, an embodiment
can be alternatively described using language "consisting
essentially of" or "consisting of."
[0020] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which this disclosure belongs.
Although methods and materials similar or equivalent to those
described herein can be used in the practice of the disclosed
methods and compositions, the exemplary methods, devices and
materials are described herein.
[0021] General texts that describe molecular biological techniques
useful herein, including the use of vectors, promoters and many
other relevant topics, include Berger and Kimmel, Guide to
Molecular Cloning Techniques, Methods in Enzymology Volume 152,
(Academic Press, Inc., San Diego, Calif.) ("Berger"); Sambrook et
al., Molecular Cloning--A Laboratory Manual, 2d ed., Vol. 1-3, Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989
("Sambrook") and Current Protocols in Molecular Biology, F. M.
Ausubel et al., eds., Current Protocols, a joint venture between
Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,
(supplemented through 1999) ("Ausubel"). Examples of protocols
sufficient to direct persons of skill through in vitro
amplification methods, including the polymerase chain reaction
(PCR), the ligase chain reaction (LCR), Q.beta.-replicase
amplification and other RNA polymerase mediated techniques (e.g.,
NASBA), e.g., for the production of the homologous nucleic acids of
the disclosure are found in Berger, Sambrook, and Ausubel, as well
as in Mullis et al. (1987) U.S. Pat. No. 4,683,202; Innis et al.,
eds. (1990) PCR Protocols: A Guide to Methods and Applications
(Academic Press Inc. San Diego, Calif.) ("Innis"); Arnheim &
Levinson (Oct. 1, 1990) C&EN 36-47; The Journal Of NIH Research
(1991) 3: 81-94; Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:
1173; Guatelli et al. (1990) Proc. Nat'l. Acad. Sci. USA 87: 1874;
Lomell et al. (1989) J. Clin. Chem 35: 1826; Landegren et al.
(1988) Science 241: 1077-1080; Van Brunt (1990) Biotechnology 8:
291-294; Wu and Wallace (1989) Gene 4:560; Barringer et al. (1990)
Gene 89:117; and Sooknanan and Malek (1995) Biotechnology 13:
563-564. Improved methods for cloning in vitro amplified nucleic
acids are described in Wallace et al., U.S. Pat. No. 5,426,039.
Improved methods for amplifying large nucleic acids by PCR are
summarized in Cheng et al. (1994) Nature 369: 684-685 and the
references cited therein, in which PCR amplicons of up to 40 kb are
generated. One of skill will appreciate that essentially any RNA
can be converted into a double stranded DNA suitable for
restriction digestion, PCR expansion and sequencing using reverse
transcriptase and a polymerase. See, e.g., Ausubel, Sambrook and
Berger, all supra.
[0022] The publications discussed throughout the text are provided
solely for their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the inventors are not entitled to antedate such disclosure by
virtue of prior disclosure.
[0023] The disclosure provides methods and compositions useful for
treating cell proliferative diseases and disorders. The disclosure
provides replication competent retroviral vectors for gene delivery
and combination therapies.
[0024] The terms "vector", "vector construct" and "expression
vector" mean the vehicle by which a DNA or RNA sequence (e.g. a
foreign gene) can be introduced into a host cell, so as to
transform the host and promote expression (e.g. transcription and
translation) of the introduced sequence. Vectors typically comprise
the DNA of a transmissible agent, into which foreign DNA encoding a
protein is inserted by restriction enzyme technology. A common type
of vector is a "plasmid", which generally is a self-contained
molecule of double-stranded DNA that can readily accept additional
(foreign) DNA and which can readily introduced into a suitable host
cell. A large number of vectors, including plasmid and fungal
vectors, have been described for replication and/or expression in a
variety of eukaryotic and prokaryotic hosts. Non-limiting examples
include pKK plasmids (Clonetech), pUC plasmids, pET plasmids
(Novagen, Inc., Madison, Wis.), pRSET or pREP plasmids (Invitrogen,
San Diego, Calif.), or pMAL plasmids (New England Biolabs, Beverly,
Mass.), and many appropriate host cells, using methods disclosed or
cited herein or otherwise known to those skilled in the relevant
art. Recombinant cloning vectors will often include one or more
replication systems for cloning or expression, one or more markers
for selection in the host, e.g., antibiotic resistance, and one or
more expression cassettes.
[0025] The terms "express" and "expression" mean allowing or
causing the information in a gene or DNA sequence to become
manifest, for example producing a protein by activating the
cellular functions involved in transcription and translation of a
corresponding gene or DNA sequence. A DNA sequence is expressed in
or by a cell to form an "expression product" such as a protein. The
expression product itself, e.g. the resulting protein, may also be
said to be "expressed" by the cell. A polynucleotide or polypeptide
is expressed recombinantly, for example, when it is expressed or
produced in a foreign host cell under the control of a foreign or
native promoter, or in a native host cell under the control of a
foreign promoter.
[0026] The disclosure provides replication competent viral vectors
that contain a heterologous polynucleotide encoding, for example, a
cytosine deaminase or mutant thereof that can be delivered to a
cell or subject. The viral vector can be an adenoviral vector, a
measles vector, a herpes vector, a retroviral vector (including a
lentiviral vector), a rhabdoviral vector such as a Vesicular
Stomatitis viral vector, a reovirus vector, a Seneca Valley Virus
vector, a poxvirus vector (including animal pox or vaccinia derived
vectors), a parvovirus vector (including an AAV vector), an
alphavirus vector or other viral vector known to one skilled in the
art (see also, e.g., Concepts in Genetic Medicine, ed. Boro
Dropulic and Barrie Carter, Wiley, 2008, Hoboken, N.J.; The
Development of Human Gene Therapy, ed. Theodore Friedmann, Cold
Springs Harbor Laboratory Press, Cold springs Harbor, N.Y., 1999;
Gene and Cell Therapy, ed. Nancy Smyth Templeton, Marcel Dekker
Inc., New York, N.Y., 2000 and Gene Therapy: Therapeutic Mechanism
and Strategies, ed. Nancy Smyth Templetone and Danilo D Lasic,
Marcel Dekker, Inc., New York, N.Y., 2000; the disclosures of which
are incorporated herein by reference).
[0027] In one embodiment, the viral vector can be a replication
competent retroviral vector capable of infecting only replicating
mammalian cells. In one embodiment, a replication competent
retroviral vector comprises an internal ribosomal entry site (IRES)
5' to the heterologous polynucleotide encoding, e.g., a cytosine
deaminase or the like. In one embodiment, the polynucleotide is 3'
to a ENV polynucleotide of a retroviral vector. In one embodiment
the viral vector is a retroviral vector capable of infecting target
cells multiple times (5 or more per diploid cell).
[0028] The disclosure also provides replication competent
retroviral vectors having increased stability relative to prior
retroviral vectors. Such increased stability during infection and
replication is important for the treatment of cell proliferative
disorders. The combination of transduction efficiency, transgene
stability and target selectivity is provided by the replication
competent retrovirus. The compositions and methods provide insert
stability and maintain transcription activity of the transgene and
the translational viability of the encoded polypeptide.
[0029] The disclosure provides modified retroviral vectors. The
modified retroviral vectors can be derived from members of the
retroviridae family. The Retroviridae family consists of three
groups: the spumaviruses-(or foamy viruses) such as the human foamy
virus (HFV); the lentiviruses, as well as visna virus of sheep; and
the oncoviruses (although not all viruses within this group are
oncogenic). The term "lentivirus" is used in its conventional sense
to describe a genus of viruses containing reverse transcriptase.
The lentiviruses include the "immunodeficiency viruses" which
include human immunodeficiency virus (HIV) type 1 and type 2 (HIV-1
and HIV-2) and simian immunodeficiency virus (SIV). The oncoviruses
have historically been further subdivided into groups A, B, C and D
on the basis of particle morphology, as seen under the electron
microscope during viral maturation. A-type particles represent the
immature particles of the B- and D-type viruses seen in the
cytoplasm of infected cells. These particles are not infectious.
B-type particles bud as mature virion from the plasma membrane by
the enveloping of intracytoplasmic A-type particles. At the
membrane they possess a toroidal core of 75 nm, from which long
glycoprotein spikes project. After budding, B-type particles
contain an eccentrically located, electron-dense core. The
prototype B-type virus is mouse mammary tumor virus (MMTV). No
intracytoplasmic particles can be observed in cells infected by
C-type viruses. Instead, mature particles bud directly from the
cell surface via a crescent `C`-shaped condensation which then
closes on itself and is enclosed by the plasma membrane. Envelope
glycoprotein spikes may be visible, along with a uniformly
electron-dense core. Budding may occur from the surface plasma
membrane or directly into intracellular vacuoles. The C-type
viruses are the most commonly studied and include many of the avian
and murine leukemia viruses (MLV). Bovine leukemia virus (BLV), and
the human T-cell leukemia viruses types I and II (HTLV-I/II) are
similarly classified as C-type particles because of the morphology
of their budding from the cell surface. However, they also have a
regular hexagonal morphology and more complex genome structures
than the prototypic C-type viruses such as the murine leukemia
viruses (MLV). D-type particles resemble B-type particles in that
they show as ring-like structures in the infected cell cytoplasm,
which bud from the cell surface, but the virion incorporate short
surface glycoprotein spikes. The electron-dense cores are also
eccentrically located within the particles. Mason Pfizer monkey
virus (MPMV) is the prototype D-type virus.
[0030] Retroviruses have been classified in various ways but the
nomenclature has been standardized in the last decade (see
ICTVdB--The Universal Virus Database, v 4 on the World Wide Web
(www) at ncbi.nlm.nih.gov/ICTVdb/ICTVdB/ and the text book
"Retroviruses" Eds Coffin, Hughs and Varmus, Cold Spring Harbor
Press 1997; the disclosures of which are incorporated herein by
reference). In one embodiment, the replication competent retroviral
vector can comprise an Orthoretrovirus or more typically a gamma
retrovirus vector.
[0031] Retroviruses are defined by the way in which they replicate
their genetic material. During replication the RNA is converted
into DNA. Following infection of the cell a double-stranded
molecule of DNA is generated from the two molecules of RNA which
are carried in the viral particle by the molecular process known as
reverse transcription. The DNA form becomes covalently integrated
in the host cell genome as a provirus, from which viral RNAs are
expressed with the aid of cellular and/or viral factors. The
expressed viral RNAs are packaged into particles and released as
infectious virion.
[0032] The retrovirus particle is composed of two identical RNA
molecules. Each wild-type genome has a positive sense,
single-stranded RNA molecule, which is capped at the 5' end and
polyadenylated at the 3' tail. The diploid virus particle contains
the two RNA strands complexed with gag proteins, viral enzymes (pol
gene products) and host tRNA molecules within a `core` structure of
gag proteins. Surrounding and protecting this capsid is a lipid
bilayer, derived from host cell membranes and containing viral
envelope (env) proteins. The env proteins bind to a cellular
receptor for the virus and the particle typically enters the host
cell via receptor-mediated endocytosis and/or membrane fusion.
[0033] After the outer envelope is shed, the viral RNA is copied
into DNA by reverse transcription. This is catalyzed by the reverse
transcriptase enzyme encoded by the pol region and uses the host
cell tRNA packaged into the virion as a primer for DNA synthesis.
In this way the RNA genome is converted into the more complex DNA
genome.
[0034] The double-stranded linear DNA produced by reverse
transcription may, or may not, have to be circularized in the
nucleus. The provirus now has two identical repeats at either end,
known as the long terminal repeats (LTR). The termini of the two
LTR sequences produces the site recognized by a pol product--the
integrase protein--which catalyzes integration, such that the
provirus is always joined to host DNA two base pairs (bp) from the
ends of the LTRs. A duplication of cellular sequences is seen at
the ends of both LTRs, reminiscent of the integration pattern of
transposable genetic elements. Retroviruses can integrate their
DNAs at many sites in host DNA, but different retroviruses have
different integration site preferences. HIV-1 and simian
immunodeficiency virus DNAs preferentially integrate into expressed
genes, murine leukemia virus (MLV) DNA preferentially integrates
near transcriptional start sites (TSSs), and avian sarcoma leukosis
virus (ASLV) and human T cell leukemia virus (HTLV) DNAs integrate
nearly randomly, showing a slight preference for genes (Derse D, et
al. (2007) Human T-cell leukemia virus type 1 integration target
sites in the human genome: comparison with those of other
retroviruses. J Virol 81:6731-6741; Lewinski M K, et al. (2006)
Retroviral DNA integration: viral and cellular determinants of
target-site selection. PLoS Pathog 2:e601).
[0035] Transcription, RNA splicing and translation of the
integrated viral DNA is mediated by host cell proteins. Variously
spliced transcripts are generated. In the case of the human
retroviruses HIV-1/2 and HTLV-I/II viral proteins are also used to
regulate gene expression. The interplay between cellular and viral
factors is a factor in the control of virus latency and the
temporal sequence in which viral genes are expressed.
[0036] Retroviruses can be transmitted horizontally and vertically.
Efficient infectious transmission of retroviruses requires the
expression on the target cell of receptors which specifically
recognize the viral envelope proteins, although viruses may use
receptor-independent, nonspecific routes of entry at low
efficiency. Normally a viral infection leads to a single or few
copies of viral genome per cell because of receptor masking or
down-regulation that in turn leads to resistance to superinfection
(Ch3 p 104 in "Retroviruses" J M Coffin, S H Hughes, & H E
Varmus 1997 Cold Spring Harbor Laboratory Press, Cold Spring Harbor
N.Y.; Fan et al. J. Virol 28:802, 1978). By manipulating the
situation in tissue culture it is possible to get some level of
multiple infection but this is typically less than 5 copies/diploid
genome. In addition, the target cell type must be able to support
all stages of the replication cycle after virus has bound and
penetrated. Vertical transmission occurs when the viral genome
becomes integrated in the germ line of the host. The provirus will
then be passed from generation to generation as though it were a
cellular gene. Hence endogenous proviruses become established which
frequently lie latent, but which can become activated when the host
is exposed to appropriate agents.
[0037] In many situations for using a recombinant replication
competent retrovirus therapeutically, it is advantageous to have
high levels of expression of the transgene that is encoded by the
recombinant replication competent retrovirus. For example, with a
prodrug activating gene such as the cytosine deaminase gene it is
advantageous to have higher levels of expression of the CD protein
in a cell so that the conversion of the prodrug 5-FC to 5-FU is
more efficient. The disclosure provides recombinant replication
competent retroviruses capable of infecting a target cell or target
cell population multiple times resulting in an average number of
copies/diploid genome of 5 or greater. Also provided are methods of
treating a cell proliferative disorder, using a recombinant
replication competent retrovirus capable of infecting a target cell
or target cell population multiple times resulting in an average
number of copies/diploid genome of 5 or greater. In further
embodiments, a combination therapy comprising thymosin-alpha-1 is
used to promote apoptosis and therapeutic effects of a RCR of the
disclosure.
[0038] As mentioned above, the integrated DNA intermediate is
referred to as a provirus. Prior gene therapy or gene delivery
systems use methods and retroviruses that require transcription of
the provirus and assembly into infectious virus while in the
presence of an appropriate helper virus or in a cell line
containing appropriate sequences enabling encapsidation without
coincident production of a contaminating helper virus. As described
below, a helper virus is not required for the production of the
recombinant retrovirus of the disclosure, since the sequences for
encapsidation are provided in the genome thus providing a
replication competent retroviral vector for gene delivery or
therapy.
[0039] Other existing replication competent retroviral vectors also
tend to be unstable and lose sequences during horizontal or
vertical transmission to an infected cell or host cell and during
replication. This may be due in-part from the presence of extra
nucleotide sequences that include repeats or which reduce the
efficiency of a polymerase.
[0040] The retroviral genome and the proviral DNA of the disclosure
have at least three genes: the gag, the pol, and the env, these
genes may be flanked by one or two long terminal (LTR) repeat, or
in the provirus are flanked by two long terminal repeat (LTR) and
sequences containing cis-acting sequences such as psi. The gag gene
encodes the internal structural (matrix, capsid, and nucleocapsid)
proteins; the pol gene encodes the RNA-directed DNA polymerase
(reverse transcriptase), protease and integrase; and the env gene
encodes viral envelope glycoproteins. The 5' and/or 3' LTRs serve
to promote transcription and polyadenylation of the virion RNAs.
The LTR contains all other cis-acting sequences necessary for viral
replication. Lentiviruses have additional genes including vif, vpr,
tat, rev, vpu, nef, and vpx (in HIV-1, HIV-2 and/or SIV).
[0041] Adjacent to the 5' LTR are sequences necessary for reverse
transcription of the genome (the tRNA primer binding site) and for
efficient encapsidation of viral RNA into particles (the Psi site).
If the sequences necessary for encapsidation (or packaging of
retroviral RNA into infectious virion) are missing from the viral
genome, the result is a cis defect which prevents encapsidation of
genomic viral RNA. This type of modified vector is what has
typically been used in prior gene delivery systems (i.e., systems
lacking elements which are required for encapsidation of the
virion) as `helper` elements providing viral proteins in trans that
package a non-replicating, but packageable, RNA genome.
[0042] In a first embodiment, the disclosure provides a recombinant
retrovirus capable of infecting a dividing cell or a cell having a
cell proliferative disorder. The recombinant replication competent
retrovirus of the disclosure comprises a polynucleotide sequence
encoding a viral GAG, a viral POL, a viral ENV, a heterologous
polynucleotide preceded by an internal ribosome entry site (IRES)
encapsulated within a virion. In one embodiment the heterologous
polynucleotide encodes a polypeptide having cytosine deaminase
activity. In yet another embodiment, a polypeptide having
thymosin-alpha-1 activity is administered simultaneously, prior to,
or after administration of the retroviral vector.
[0043] The phrase "non-dividing" cell refers to a cell that does
not go through mitosis. Non-dividing cells may be blocked at any
point in the cell cycle, (e.g., G.sub.0/G.sub.1, G.sub.1/S,
G.sub.2/M), as long as the cell is not actively dividing. For ex
vivo infection, a dividing cell can be treated to block cell
division by standard techniques used by those of skill in the art,
including, irradiation, aphidocolin treatment, serum starvation,
and contact inhibition. However, it should be understood that ex
vivo infection is often performed without blocking the cells since
many cells are already arrested (e.g., stem cells). For example, a
recombinant lentivirus vector is capable of infecting non-dividing
cells. Examples of pre-existing non-dividing cells in the body
include neuronal, muscle, liver, skin, heart, lung, and bone marrow
cells, and their derivatives. For dividing cells onco-retroviral
vectors can be used.
[0044] By "dividing" cell is meant a cell that undergoes active
mitosis, or meiosis. Such dividing cells include stem cells, skin
cells (e.g., fibroblasts and keratinocytes), gametes, and other
dividing cells known in the art. Of particular interest and
encompassed by the term dividing cell are cells having cell
proliferative disorders, such as neoplastic cells. The term "cell
proliferative disorder" refers to a condition characterized by an
abnormal number of cells. The condition can include both
hypertrophic (the continual multiplication of cells resulting in an
overgrowth of a cell population within a tissue) and hypotrophic (a
lack or deficiency of cells within a tissue) cell growth or an
excessive influx or migration of cells into an area of a body. The
cell populations are not necessarily transformed, tumorigenic or
malignant cells, but can include normal cells as well. Cell
proliferative disorders include disorders associated with an
overgrowth of connective tissues, such as various fibrotic
conditions, including scleroderma, arthritis and liver cirrhosis.
Cell proliferative disorders include neoplastic disorders such as
head and neck carcinomas. Head and neck carcinomas would include,
for example, carcinoma of the mouth, esophagus, throat, larynx,
thyroid gland, tongue, lips, salivary glands, nose, paranasal
sinuses, nasopharynx, superior nasal vault and sinus tumors,
esthesioneuroblastoma, squamous call cancer, malignant melanoma,
sinonasal undifferentiated carcinoma (SNUC), brain (including
glioblastomas) or blood neoplasia. Also included are carcinoma's of
the regional lymph nodes including cervical lymph nodes,
prelaryngeal lymph nodes, pulmonary juxtaesophageal lymph nodes and
submandibular lymph nodes (Harrison's Principles of Internal
Medicine (eds., Isselbacher, et al., McGraw-Hill, Inc., 13th
Edition, pp 1850-1853, 1994). Other cancer types, include, but are
not limited to, lung cancer, colon-rectum cancer, breast cancer,
prostate cancer, urinary tract cancer, uterine cancer lymphoma,
oral cancer, pancreatic cancer, leukemia, melanoma, stomach cancer,
skin cancer and ovarian cancer. The cell proliferative disease also
includes rheumatoid arthritis (O'Dell NEJM 350:2591 2004) and other
auto-immune disorders (Mackay et al NEJM 345:340 2001) that are
often characterized by inappropriate proliferation of cells of the
immune system.
[0045] The heterologous nucleic acid sequence is operably linked to
an IRES. As used herein, the term "heterologous" nucleic acid
sequence or transgene refers to (i) a sequence that does not
normally exist in a wild-type retrovirus, (ii) a sequence that
originates from a foreign species, or (iii) if from the same
species, it may be substantially modified from its original form.
Alternatively, an unchanged nucleic acid sequence that is not
normally expressed in a cell is a heterologous nucleic acid
sequence.
[0046] Depending upon the intended use of the retroviral vector of
the disclosure any number of heterologous polynucleotide or nucleic
acid sequences may be inserted into the retroviral vector. For
example, for in vitro studies commonly used marker genes or
reporter genes may be used, including, antibiotic resistance and
fluorescent molecules (e.g., GFP). Additional polynucleotide
sequences encoding any desired polypeptide sequence may also be
inserted into the vector of the disclosure. Where in vivo delivery
of a heterologous nucleic acid sequence is sought both therapeutic
and non-therapeutic sequences may be used. For example, the
heterologous sequence can encode a therapeutic molecule including
antisense molecules (miRNA, siRNA) or ribozymes directed to a
particular gene associated with a cell proliferative disorder or
other gene-associated disease or disorder, the heterologous
sequence can be a suicide gene (e.g., HSV-tk or PNP or cytosine
deaminase; either modified or unmodified), a growth factor or a
therapeutic protein (e.g., Factor IX, IL2, and the like). Other
therapeutic proteins applicable to the disclosure are easily
identified in the art.
[0047] In one embodiment, the heterologous polynucleotide within
the vector comprises a cytosine deaminase that has been optimized
for expression in a human cell. In a further embodiment, the
cytosine deaminase comprises a sequence that has been human codon
optimized and comprises mutations that increase the cytosine
deaminase's stability (e.g., reduced degradation or increased
thermo-stability) compared to a wild-type cytosine deaminase. In
yet another embodiment, the heterologous polynucleotide encodes a
fusion construct comprising a cytosine deaminase (either human
codon optimized or non-optimized, either mutated or non-mutated)
operably linked to a polynucleotide encoding a polypeptide having
UPRT or OPRT activity. In another embodiment, the heterologous
polynucleotide comprises a CD polynucleotide of the disclosure
(e.g., SEQ ID NO:3, 5, 11, 13, 15, or 17).
[0048] In another embodiment, replication competent retroviral
vector can comprise a heterologous polynucleotide encoding a
polypeptide comprising a cytosine deaminase (as described herein)
and may further comprise a polynucleotide comprising a miRNA or
siRNA molecule either as part of the primary transcript from the
viral promoter or linked to a promoter, which can be cell-type or
tissue specific.
[0049] For examples, miRNAs that are down-regulated in cancers
could be useful as anticancer agents. Examples include mir-128-1,
let-7, miR-26, miR-124, and miR-137 (Esquela-Kerscher et al., 2008
Cell Cycle 7, 759-764; Kumar et al., 2008 Proc Natl Acad Sci USA
105, 3903-3908; Kota et al., 2009 Cell 137, 1005-1017; Silber et
al., 2008 BMC Medicine 6:14 1-17). miR-128 expression has reported
to be enriched in the central nervous system and has been observed
to be down-regulated in glioblastomas (Sempere et al., 2004 Genome
Biology 5:R13.5-11; Godlewski et al., 2008 Cancer Res 68: (22)
9125-9130). miR-128 is encoded by two distinct genes, miR-128-1 and
miR-128-2. Both are processed into identical mature sequence. Bmi-1
and E2F3a have been reported to be the direct targets of miR-128
(Godlewski et al., 2008 Cancer Res 68: (22) 9125-9130; Zhang et
al., 2009 J. Mol Med 87:43-51). In addition, Bmi-1 expression has
been observed to be up-regulated in a variety of human cancers,
including gliomas, mantle cell lymphomas, non-small cell lung
cancer B-cell non-Hodgkin's lymphoma, breast, colorectal and
prostate cancer. Furthermore, Bmi-1 has been demonstrated to be
required for the self-renewal of stem cells from diverse tissues,
including neuronal stem cells as well as "stem-like" cell
population in gliomas.
[0050] In one embodiment, the disclosure provides a recombinant
replication competent retroviral vector that contains a single copy
of the miR-142-3p target sequence (142-3pT, SEQ ID NO:35)
downstream of the transgene, such as yCD2 or GFP, linked to the
IRES. In addition to miR181 and miR-223, the target sequence of
other tissue or cell-enriched miRNA can be incorporated into the
vector to restrict viral spread in specific tissue or cell type
manner. For example, miR-133 and miR206 expressions are highly
enriched in muscle cells (Kelly et al., 2008 Nature Medicine 14:11
1278-1283.
[0051] In another embodiment, the disclosure provides a recombinant
replication competent retroviral vector that contains 4 copies of
the 142-3pT (SEQ ID NO: 36) downstream of the transgene, such as
yCD2 or GFP, linked to the IRES. In addition to miR181 and miR-223,
the target sequence of other tissue or cell-enriched miRNA can be
incorporated into the vector to restrict viral spread in specific
tissue or cell type manner.
[0052] In yet further embodiments, the heterologous polynucleotide
may comprise a cytokine such as an interleukin, interferon gamma or
the like. Cytokines that may expressed from a retroviral vector of
the disclosure include, but are not limited to, IL-1alpha,
IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,
IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19,
IL-20, and IL-21, anti-CD40, CD40L, IFN-gamma and TNF-alpha,
soluble forms of TNF-alpha, lymphotoxin-alpha (LT-alpha, also known
as TNF-beta), LT-beta (found in complex heterotrimer
LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3,
OX40L, TNF-gamma (International Publication No. WO 96/14328), AIM-I
(International Publication No. WO 97/33899), endokine-alpha
(International Publication No. WO 98/07880), OPG, and
neutrokine-alpha (International Publication No. WO 98/18921, OX40,
and nerve growth factor (NGF), and soluble forms of Fas, CD30,
CD27, CD40 and 4-IBB, TR2 (International Publication No. WO
96/34095), DR3 (International Publication No. WO 97/33904), DR4
(International Publication No. WO 98/32856), TR5 (International
Publication No. WO 98/30693), TRANK, TR9 (International Publication
No. WO 98/56892), TR10 (International Publication No. WO 98/54202),
312C2 (International Publication No. WO 98/06842), and TR12, and
soluble forms CD154, CD70, and CD153. Angiogenic proteins may be
useful in some embodiments, particularly for protein production
from cell lines. Such angiogenic factors include, but are not
limited to, Glioma Derived Growth Factor (GDGF), Platelet Derived
Growth Factor-A (PDGF-A), Platelet Derived Growth Factor-B
(PDGF-B), Placental Growth Factor (PIGF), Placental Growth Factor-2
(PIGF-2), Vascular Endothelial Growth Factor (VEGF), Vascular
Endothelial Growth Factor-A (VEGF-A), Vascular Endothelial Growth
Factor-2 (VEGF-2), Vascular Endothelial Growth Factor B (VEGF-3),
Vascular Endothelial Growth Factor B-1 86 (VEGF-B186), Vascular
Endothelial Growth Factor-D (VEGF-D), Vascular Endothelial Growth
Factor-D (VEGF-D), and Vascular Endothelial Growth Factor-E
(VEGF-E). Fibroblast Growth Factors may be delivered by a vector of
the disclosure and include, but are not limited to, FGF-1, FGF-2,
FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11,
FGF-12, FGF-13, FGF-14, and FGF-15. Hematopoietic growth factors
may be delivered using vectors of the disclosure, such growth
factors include, but are not limited to, granulocyte macrophage
colony stimulating factor (GM-CSF) (sargramostim), granulocyte
colony stimulating factor (G-CSF) (filgrastim), macrophage colony
stimulating factor (M-CSF, CSF-1) erythropoietin (epoetin alfa),
stem cell factor (SCF, c-kit ligand, steel factor), megakaryocyte
colony stimulating factor, PIXY321 (a GMCSF/IL-3) fusion protein
and the like.
[0053] The term "regulatory nucleic acid sequence" refers
collectively to promoter sequences, polyadenylation signals,
transcription termination sequences, upstream regulatory domains,
origins of replication, enhancers and the like, which collectively
provide for the replication, transcription and translation of a
coding sequence in a recipient cell. Not all of these control
sequences need always be present so long as the selected coding
sequence is capable of being replicated, transcribed and translated
in an appropriate host cell. One skilled in the art can readily
identify regulatory nucleic acid sequence from public databases and
materials. Furthermore, one skilled in the art can identify a
regulatory sequence that is applicable for the intended use, for
example, in vivo, ex vivo, or in vitro.
[0054] An internal ribosome entry sites ("IRES") refers to a
segment of nucleic acid that promotes the entry or retention of a
ribosome during translation of a coding sequence usually 3' to the
IRES. In some embodiments the IRES may comprise a splice
acceptor/donor site, however, preferred IRESs lack a splice
acceptor/donor site. Normally, the entry of ribosomes into
messenger RNA takes place via the cap located at the 5' end of all
eukaryotic mRNAs. However, there are exceptions to this universal
rule. The absence of a cap in some viral mRNAs suggests the
existence of alternative structures permitting the entry of
ribosomes at an internal site of these RNAs. To date, a number of
these structures, designated IRES on account of their function,
have been identified in the 5' noncoding region of uncapped viral
mRNAs, such as that, in particular, of picornaviruses such as the
poliomyelitis virus (Pelletier et al., 1988, Mol. Cell. Biol., 8,
1103-1112) and the EMCV virus (encephalo-myocarditis virus (Jang et
al., J. Virol., 1988, 62, 2636-2643). The disclosure provides the
use of an IRES in the context of a replication-competent retroviral
vector.
[0055] The term "promoter region" is used herein in its ordinary
sense to refer to a nucleotide region comprising a DNA regulatory
sequence, wherein the regulatory sequence is derived from a gene
which is capable of binding RNA polymerase and initiating
transcription of a downstream (3'-direction) coding sequence. The
regulatory sequence may be homologous or heterologous to the
desired gene sequence. For example, a wide range of promoters may
be utilized, including viral or mammalian promoter as described
above.
[0056] The heterologous nucleic acid sequence is typically under
control of either the viral LTR promoter-enhancer signals or an
internal promoter, and retained signals within the retroviral LTR
can still bring about efficient integration of the vector into the
host cell genome. Accordingly, the recombinant retroviral vectors
of the disclosure, the desired sequences, genes and/or gene
fragments can be inserted at several sites and under different
regulatory sequences. For example, a site for insertion can be the
viral enhancer/promoter proximal site (i.e., 5' LTR-driven gene
locus). Alternatively, the desired sequences can be inserted into a
regulatory sequence distal site (e.g., the IRES sequence 3' to the
env gene) or where two or more heterologous sequences are present
one heterologous sequence may be under the control of a first
regulatory region and a second heterologous sequence under the
control of a second regulatory region. Other distal sites include
viral promoter sequences, where the expression of the desired
sequence or sequences is through splicing of the promoter proximal
cistron, an internal heterologous promoter as SV40 or CMV, or an
internal ribosome entry site (IRES) can be used.
[0057] In one embodiment, the retroviral genome of the disclosure
contains an IRES comprising a cloning site downstream of the IRES
for insertion of a desired/heterologous polynucleotide. In one
embodiment, the IRES is located 3' to the env gene in the
retroviral vector, but 5' to the desired heterologous
polynucleotide. Accordingly, a heterologous polynucleotide encoding
a desired polypeptide may be operably linked to the IRES.
[0058] In another embodiment, a targeting polynucleotide sequence
is included as part of the recombinant retroviral vector of the
disclosure. The targeting polynucleotide sequence is a targeting
ligand (e.g., peptide hormones such as heregulin, a single-chain
antibodies, a receptor or a ligand for a receptor), a
tissue-specific or cell-type specific regulatory element (e.g., a
tissue-specific or cell-type specific promoter or enhancer), or a
combination of a targeting ligand and a tissue-specific/cell-type
specific regulatory element. Preferably, the targeting ligand is
operably linked to the env protein of the retrovirus, creating a
chimeric retroviral env protein. The viral GAG, viral POL and viral
ENV proteins can be derived from any suitable retrovirus (e.g., MLV
or lentivirus-derived). In another embodiment, the viral ENV
protein is non-retrovirus-derived (e.g., CMV or VSV).
[0059] In one embodiment, the recombinant retrovirus of the
disclosure is genetically modified in such a way that the virus is
targeted to a particular cell type (e.g., smooth muscle cells,
hepatic cells, renal cells, fibroblasts, keratinocytes, mesenchymal
stem cells, bone marrow cells, chondrocyte, epithelial cells,
intestinal cells, mammary cells, neoplastic cells, glioma cells,
neuronal cells and others known in the art) such that the
recombinant genome of the retroviral vector is delivered to a
target non-dividing, a target dividing cell, or a target cell
having a cell proliferative disorder.
[0060] In one embodiment, the retroviral vector is targeted to the
cell by binding to cells having a molecule on the external surface
of the cell. This method of targeting the retrovirus utilizes
expression of a targeting ligand on the coat of the retrovirus to
assist in targeting the virus to cells or tissues that have a
receptor or binding molecule which interacts with the targeting
ligand on the surface of the retrovirus. After infection of a cell
by the virus, the virus injects its nucleic acid into the cell and
the retrovirus genetic material can integrate into the host cell
genome.
[0061] In another embodiment, targeting uses cell- or
tissue-specific regulatory elements to promote expression and
transcription of the viral genome in a targeted cell which actively
utilizes the regulatory elements, as described more fully below.
The transferred retrovirus genetic material is then transcribed and
translated into proteins within the host cell. The targeting
regulatory element is typically linked to the 5' and/or 3' LTR,
creating a chimeric LTR.
[0062] By inserting a heterologous polynucleotide of interest into
the viral vector of the disclosure, along with another gene which
encodes, for example, the ligand for a receptor on a specific
target cell, the vector is now target specific. Viral vectors can
be made target specific by attaching, for example, a sugar, a
glycolipid, or a protein. Targeting can be accomplished by using an
antibody to target the viral vector. Those of skill in the art will
know of, or can readily ascertain, specific polynucleotide
sequences which can be inserted into the viral genome or proteins
which can be attached to a viral envelope to allow target specific
delivery of the viral vector containing the nucleic acid sequence
of interest.
[0063] Thus, the disclosure includes in one embodiment, a chimeric
env protein comprising a retroviral ENV protein operably linked to
a targeting polypeptide. The targeting polypeptide can be a cell
specific receptor molecule, a ligand for a cell specific receptor,
an antibody or antibody fragment to a cell specific antigenic
epitope or any other ligand easily identified in the art which is
capable of binding or interacting with a target cell. Examples of
targeting polypeptides or molecules include bivalent antibodies
using biotin-streptavidin as linkers (Etienne-Julan et al., J. Of
General Virol., 73, 3251-3255 (1992); Roux et al., Proc. Natl.
Acad. Sci USA 86, 9079-9083 (1989)), recombinant virus containing
in its envelope a sequence encoding a single-chain antibody
variable region against a hapten (Russell et al., Nucleic Acids
Research, 21, 1081-1085 (1993)), cloning of peptide hormone ligands
into the retrovirus envelope (Kasahara et al., Science, 266,
1373-1376 (1994)), chimeric EPO/env constructs (Kasahara et al.,
1994), single-chain antibody against the low density lipoprotein
(LDL) receptor in the ecotropic MLV envelope, resulting in specific
infection of HeLa cells expressing LDL receptor (Somia et al.,
Proc. Natl. Acad. Sci USA, 92, 7570-7574 (1995)), similarly the
host range of ALV can be altered by incorporation of an integrin
ligand, enabling the virus to now cross species to specifically
infect rat glioblastoma cells (Valsesia-Wittmann et al., J. Virol.
68, 4609-4619 (1994)), and Dornberg and co-workers (Chu and
Dornburg, J. Virol 69, 2659-2663 (1995); M. Engelstadter et al.
Gene Therapy 8, 1202-1206 (2001)) have reported tissue-specific
targeting of spleen necrosis virus (SNV), an avian retrovirus,
using envelopes containing single-chain antibodies directed against
tumor markers.
[0064] The disclosure provides a method of producing a recombinant
retrovirus capable of infecting a target cell comprising
transfecting a suitable host cell with the following: a vector
comprising a polynucleotide sequence encoding a viral gag, a viral
pol and a viral env, and a heterologous polynucleotide, operably
linked to a regulatory nucleic acid sequence, and recovering the
recombinant virus.
[0065] The retrovirus and methods of the disclosure provide a
replication competent retrovirus that does not require helper virus
or additional nucleic acid sequence or proteins in order to
propagate and produce virion. For example, the nucleic acid
sequences of the retrovirus of the disclosure encode a group
specific antigen and reverse transcriptase, (and integrase and
protease-enzymes necessary for maturation and reverse
transcription), respectively, as discussed above. The viral gag and
pol can be derived from a lentivirus, such as HIV or an oncovirus
or gammaretrovirus such as MoMLV. In addition, the nucleic acid
genome of the retrovirus of the disclosure includes a sequence
encoding a viral envelope (ENV) protein. The env gene can be
derived from any retroviruses. The env may be an amphotropic
envelope protein which allows transduction of cells of human and
other species, or may be an ecotropic envelope protein, which is
able to transduce only mouse and rat cells. Further, it may be
desirable to target the recombinant virus by linkage of the
envelope protein with an antibody or a particular ligand for
targeting to a receptor of a particular cell-type. As mentioned
above, retroviral vectors can be made target specific by inserting,
for example, a glycolipid, or a protein. Targeting is often
accomplished by using an antibody to target the retroviral vector
to an antigen on a particular cell-type (e.g., a cell type found in
a certain tissue, or a cancer cell type). Those of skill in the art
will know of, or can readily ascertain without undue
experimentation, specific methods to achieve delivery of a
retroviral vector to a specific target. In one embodiment, the env
gene is derived from a non-retrovirus (e.g., CMV or VSV). Examples
of retroviral-derived env genes include, but are not limited to:
Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus
(HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia
virus (GaLV), human immunodeficiency virus (HIV) and Rous Sarcoma
Virus (RSV). Other env genes such as Vesicular stomatitis virus
(VSV) (Protein G), cytomegalovirus envelope (CMV), or influenza
virus hemagglutinin (HA) can also be used.
[0066] In one embodiment, the retroviral genome is derived from an
onco-retrovirus, and more particularly a mammalian onco-retrovirus.
In a further embodiment, the retroviral genome is derived from a
gamma retrovirus, and more particularly a mammalian gamma
retrovirus. By "derived" is meant that the parent polynucleotide
sequence is an wild-type oncovirus which has been modified by
insertion or removal of naturally occurring sequences (e.g.,
insertion of an IRES, insertion of a heterologous polynucleotide
encoding a polypeptide or inhibitory nucleic acid of interest,
swapping of a more effective promoter from a different retrovirus
or virus in place of the wild-type promoter and the like).
[0067] In another embodiment, the disclosure provides retroviral
vectors that are targeted using regulatory sequences. Cell- or
tissue-specific regulatory sequences (e.g., promoters) can be
utilized to target expression of gene sequences in specific cell
populations. Suitable mammalian and viral promoters for the
disclosure are described elsewhere herein. Accordingly, in one
embodiment, the disclosure provides a retrovirus having
tissue-specific promoter elements at the 5' end of the retroviral
genome. Typically, the tissue-specific regulatory
elements/sequences are in the U3 region of the LTR of the
retroviral genome, including for example cell- or tissue-specific
promoters and enhancers to neoplastic cells (e.g., tumor
cell-specific enhancers and promoters), and inducible promoters
(e.g., tetracycline).
[0068] Transcription control sequences of the disclosure can also
include naturally occurring transcription control sequences
naturally associated with a gene encoding a superantigen, a
cytokine or a chemokine.
[0069] In some circumstances, it may be desirable to regulate
expression. For example, different viral promoters with varying
strengths of activity may be utilized depending on the level of
expression desired. In mammalian cells, the CMV immediate early
promoter if often used to provide strong transcriptional
activation. Modified versions of the CMV promoter that are less
potent have also been used when reduced levels of expression of the
transgene are desired. When expression of a transgene in
hematopoietic cells is desired, retroviral promoters such as the
LTRs from MLV or MMTV can be used. Other viral promoters that can
be used include SV40, RSV LTR, HIV-1 and HIV-2 LTR, adenovirus
promoters such as from the E1A, E2A, or MLP region, AAV LTR,
cauliflower mosaic virus, HSV-TK, and avian sarcoma virus.
[0070] Similarly tissue specific or selective promoters may be used
to effect transcription in specific tissues or cells so as to
reduce potential toxicity or undesirable effects to non-targeted
tissues. For example, promoters such as the PSA, probasin,
prostatic acid phosphatase or prostate-specific glandular
kallikrein (hK2) may be used to target gene expression in the
prostate. The Whey accessory protein (WAP) may be used for breast
tissue expression (Andres et al., PNAS 84:1299-1303, 1987). Other
promoters/regulatory domains that can be used are set forth in
Table 1.
[0071] "Tissue-specific regulatory elements" are regulatory
elements (e.g., promoters) that are capable of driving
transcription of a gene in one tissue while remaining largely
"silent" in other tissue types. It will be understood, however,
that tissue-specific promoters may have a detectable amount of
"background" or "base" activity in those tissues where they are
silent. The degree to which a promoter is selectively activated in
a target tissue can be expressed as a selectivity ratio (activity
in a target tissue/activity in a control tissue). In this regard, a
tissue specific promoter useful in the practice of the disclosure
typically has a selectivity ratio of greater than about 5.
Preferably, the selectivity ratio is greater than about 15.
[0072] In certain indications, it may be desirable to activate
transcription at specific times after administration of the
recombinant replication competent retrovirus of the disclosure
(RRCR). This may be done with promoters that are hormone or
cytokine regulatable. For example in therapeutic applications where
the indication is a gonadal tissue where specific steroids are
produced or routed to, use of androgen or estrogen regulated
promoters may be advantageous. Such promoters that are hormone
regulatable include MMTV, MT-1, ecdysone and RuBisco. Other hormone
regulated promoters such as those responsive to thyroid, pituitary
and adrenal hormones may be used. Cytokine and inflammatory protein
responsive promoters that could be used include K and T Kininogen
(Kageyama et al., 1987), c-fos, TNF-alpha, C-reactive protein
(Arcone et al., 1988), haptoglobin (Oliviero et al., 1987), serum
amyloid A2, C/EBP alpha, IL-1, IL-6 (Poli and Cortese, 1989),
Complement C3 (Wilson et al., 1990), IL-8, alpha-1 acid
glycoprotein (Prowse and Baumann, 1988), alpha-1 antitrypsin,
lipoprotein lipase (Zechner et al., 1988), angiotensinogen (Ron et
al., 1990), fibrinogen, c-jun (inducible by phorbol esters,
TNF-alpha, UV radiation, retinoic acid, and hydrogen peroxide),
collagenase (induced by phorbol esters and retinoic acid),
metallothionein (heavy metal and glucocorticoid inducible),
Stromelysin (inducible by phorbol ester, interleukin-1 and EGF),
alpha-2 macroglobulin and alpha-1 antichymotrypsin. Tumor specific
promoters such as osteocalcin, hypoxia-responsive element (HRE),
MAGE-4, CEA, alpha-fetoprotein, GRP78/BiP and tyrosinase may also
be used to regulate gene expression in tumor cells.
[0073] In addition, this list of promoters should not be construed
to be exhaustive or limiting, those of skill in the art will know
of other promoters that may be used in conjunction with the
promoters and methods disclosed herein.
TABLE-US-00001 TABLE 1 TISSUE SPECIFIC PROMOTERS Tissue Promoter
Pancreas Insulin Elastin Amylase pdr-1 pdx-1 glucokinase Liver
Albumin PEPCK HBV enhancer .alpha. fetoprotein apolipoprotein C
.alpha.-1 antitrypsin vitellogenin, NF-AB Transthyretin Skeletal
muscle Myosin H chain Muscle creatine kinase Dystrophin Calpain p94
Skeletal alpha-actin fast troponin 1 Skin Keratin K6 Keratin K1
Lung CFTR Human cytokeratin 18 (K18) Pulmonary surfactant proteins
A, B and C CC-10 P1 Smooth muscle sm22 .alpha. SM-alpha-actin
Endothelium Endothelin-1 E-selectin von Willebrand factor TIE
(Korhonen et al., 1995) KDR/flk-1 Melanocytes Tyrosinase Adipose
tissue Lipoprotein lipase (Zechner et al., 1988) Adipsin
(Spiegelman et al. , 1989) acetyl-CoA carboxylase (Pape and Kim,
1989) glycerophosphate dehydrogenase (Dani et al., 1989) adipocyte
P2 (Hunt et al., 1986) Breast Whey Acidic Protein (WAP) (Andres et
al. PNAS 84: 1299-1303 1987 Blood .beta.-globin
[0074] It will be further understood that certain promoters, while
not restricted in activity to a single tissue type, may
nevertheless show selectivity in that they may be active in one
group of tissues, and less active or silent in another group. Such
promoters are also termed "tissue specific", and are contemplated
for use with the disclosure. For example, promoters that are active
in a variety of central nervous system (CNS) neurons may be
therapeutically useful in protecting against damage due to stroke,
which may affect any of a number of different regions of the brain.
Accordingly, the tissue-specific regulatory elements used in the
disclosure, have applicability to regulation of the heterologous
proteins as well as applicability as a targeting polynucleotide
sequence in the present retroviral vectors.
[0075] In yet another embodiment, the disclosure provides plasmids
comprising a recombinant retroviral derived construct. The plasmid
can be directly introduced into a target cell or a cell culture
such as NIH 3T3 or other tissue culture cells. The resulting cells
release the retroviral vector into the culture medium.
[0076] The disclosure provides a polynucleotide construct
comprising from 5' to 3': a promoter or regulatory region useful
for initiating transcription; a psi packaging signal; a gag
encoding nucleic acid sequence, a pol encoding nucleic acid
sequence; an env encoding nucleic acid sequence; an internal
ribosome entry site nucleic acid sequence; a heterologous
polynucleotide encoding a marker, therapeutic or diagnostic
polypeptide; and a LTR nucleic acid sequence. As described
elsewhere herein and as follows the various segment of the
polynucleotide construct of the disclosure (e.g., a recombinant
replication competent retroviral polynucleotide) are engineered
depending in part upon the desired host cell, expression timing or
amount, and the heterologous polynucleotide. A replication
competent retroviral construct of the disclosure (e.g., comprising
SEQ ID NO:19, 20 or 22) can be divided up into a number of domains
that may be individually modified by those of skill in the art.
[0077] For example, the promoter can comprise a CMV promoter having
a sequence as set forth in SEQ ID NO:19, 20 or 22 from nucleotide 1
to about nucleotide 582 and may include modification to one or more
(e.g., 2-5, 5-10, 10-20, 20-30, 30-50, 50-100 or more nucleic acid
bases) so long as the modified promoter is capable of directing and
initiating transcription. In one embodiment, the promoter or
regulatory region comprises a CMV-R-U5 domain polynucleotide. The
CMV-R-U5 domain comprises the immediately early promoter from human
cytomegalovirus to the MLV R-U5 region. In one embodiment, the
CMV-R-U5 domain polynucleotide comprises a sequence as set forth in
SEQ ID NO:19, 20 or 22 from about nucleotide 1 to about nucleotide
1202 or sequences that are at least 95% identical to a sequence as
set forth in SEQ ID NO:19, 20, or 22 wherein the polynucleotide
promotes transcription of a nucleic acid molecule operably linked
thereto. The gag domain of the polynucleotide may be derived from
any number of retroviruses, but will typically be derived from an
oncoretrovirus and more particularly from a mammalian
oncoretrovirus. In one embodiment the gag domain comprises a
sequence from about nucleotide number 1203 to about nucleotide 2819
or a sequence having at least 95%, 98%, 99% or 99.8% (rounded to
the nearest 10.sup.th) identity thereto. The pol domain of the
polynucleotide may be derived from any number of retroviruses, but
will typically be derived from an oncoretrovirus and more
particularly from a mammalian oncoretrovirus. In one embodiment the
pol domain comprises a sequence from about nucleotide number 2820
to about nucleotide 6358 or a sequence having at least 95%, 98%,
99% or 99.9% (roundest to the nearest 10.sup.th) identity thereto.
The env domain of the polynucleotide may be derived from any number
of retroviruses, but will typically be derived from an
oncoretrovirus or gamma-retrovirus and more particularly from a
mammalian oncoretrovirus or gamma-retrovirus. In some embodiments
the env coding domain comprises an amphotropic env domain. In one
embodiment the env domain comprises a sequence from about
nucleotide number 6359 to about nucleotide 8323 or a sequence
having at least 95%, 98%, 99% or 99.8% (roundest to the nearest
10.sup.th) identity thereto. The IRES domain of the polynucleotide
may be obtained from any number of internal ribosome entry sites.
In one embodiment, IRES is derived from an encephalomyocarditis
virus. In one embodiment the IRES domain comprises a sequence from
about nucleotide number 8327 to about nucleotide 8876 or a sequence
having at least 95%, 98%, or 99% (roundest to the nearest
10.sup.th) identity thereto so long as the domain allows for entry
of a ribosome. The heterologous domain can comprise a cytosine
deaminase of the disclosure. In one embodiment, the CD
polynucleotide comprises a human codon optimized sequence. In yet
another embodiment, the CD polynucleotide encodes a mutant
polypeptide having cytosine deaminase, wherein the mutations confer
increased thermal stabilization that increase the melting
temperature (T.sub.m) by 10.degree. C. allowing sustained kinetic
activity over a broader temperature range and increased accumulated
levels of protein. In one embodiment, the cytosine deaminase
comprises a sequence as set forth in SEQ ID NO:19 or 22 from about
nucleotide number 8877 to about 9353. The heterologous domain may
be followed by a polypurine rich domain. The 3' LTR can be derived
from any number of retroviruses, typically an oncoretrovirus and
preferably a mammalian oncoretrovirus. In one embodiment, the 3'
LTR comprises a U3-R-U5 domain. In yet another embodiment the LTR
comprises a sequence as set forth in SEQ ID NO:19 or 22 from about
nucleotide 9405 to about 9998 or a sequence that is at least 95%,
98% or 99.5% (rounded to the nearest 10.sup.th) identical
thereto.
[0078] The disclosure also provides a recombinant retroviral vector
comprising from 5' to 3' a CMV-R-U5, fusion of the immediate early
promoter from human cytomegalovirus to the MLV R-U5 region; a PBS,
primer binding site for reverse transcriptase; a 5' splice site; a
psi (.psi.) packaging signal; a gag, ORF for MLV group specific
antigen; a pol, ORF for MLV polymerase polyprotein; a 3' splice
site; a 4070A env, ORF for envelope protein of MLV strain 4070A; an
IRES, internal ribosome entry site of encephalomyocarditis virus; a
modified cytosine deaminase (thermostablized and codon optimized);
a PPT, polypurine tract; and a U3-R-U5, MLV long terminal repeat.
This structure is further depicted in FIG. 3.
[0079] The disclosure also provides a retroviral vector comprising
a sequence as set forth in SEQ ID NO:19, 20 or 22.
[0080] The retroviral vectors can be used to treat a wide range of
disease and disorders including a number of cell proliferative
diseases and disorders (see, e.g., U.S. Pat. Nos. 4,405,712 and
4,650,764; Friedmann, 1989, Science, 244:1275-1281; Mulligan, 1993,
Science, 260:926-932, R. Crystal, 1995, Science 270:404-410, each
of which are incorporated herein by reference in their entirety,
see also, The Development of Human Gene Therapy, Theodore
Friedmann, Ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1999. ISBN 0-87969-528-5, which is incorporated
herein by reference in its entirety).
[0081] The disclosure also provides gene therapy for the treatment
of cell proliferative disorders. Such therapy would achieve its
therapeutic effect by introduction of an appropriate therapeutic
polynucleotide (e.g., antisense, ribozymes, suicide genes, siRNA),
into cells of subject having the proliferative disorder. Delivery
of polynucleotide constructs can be achieved using the recombinant
retroviral vector of the disclosure, particularly if it is based on
MLV, which is capable of infecting dividing cells.
[0082] In addition, the therapeutic methods (e.g., the gene therapy
or gene delivery methods) as described herein can be performed in
vivo or ex vivo. It may be preferable to remove the majority of a
tumor prior to gene therapy, for example surgically or by
radiation. In some aspects, the retroviral therapy may be preceded
or followed by surgery, chemotherapy or radiation therapy.
[0083] The methods and compositions of the disclosure are useful in
combination therapies including therapies with bevacizumab. As
described herein a replication competent retrovirus (RCR) of the
disclosure comprising a therapeutic (e.g., a cytotoxic gene) is
useful in treating cell proliferative disorders. An advantage of
the RCR of the disclosure includes its ability to infect
replicating cells cancer cells. Where the transgene of the vector
comprises a cytotoxic gene (e.g., a gene that encodes a polypeptide
that converts a non-cytotoxic agent to a cytotoxic agent) provides
the ability to kill cancer cells.
[0084] In another embodiment, the methods and composition of the
disclosure are useful in combination with agents that promote
apoptosis or that modify expression of cytokines or agents that
promote apoptosis. For example, a retroviral vector of the
disclosure comprising a polynucleotide encoding a polypeptide
having cytosine deaminase activity can be administered prior to,
simultaneously with, or after administration of a peptide or
polypeptide having thymosin-alpha-1 activity. In one embodiment,
the thymosin-alpha-1 polypeptide is administered at about 0.1-16
mg/kg.
[0085] Thymosin alpha-1 (Zadaxin.TM.) functions by increasing the
sensitivity of neoplastic cells to chemotherapeutic agents by
upregulating pro-apoptotic proteins. Specifically, Thymosin alpha-1
upregulates pro-apoptotic FasL, FasR and TNFalpha-R1. In
combination with a RCR of the disclosure, Thymosin alpha-1
functions as an adjuvant to increase the sensitivity of neoplastic
cells to 5-FU thereby increasing the effectiveness of Toca 511 5-FC
to 5-FU conversion as a chemotherapeutic agent after administration
of RCR derived from T5.0002 and known as Toca 511. Thymosin alpha-1
can also function as an immunomodulatory agent increasing the
recruitment and activity of immune components thereby leading to
enhancement of vaccine effectiveness of RRV therapy.
[0086] A polypeptide having thymosin-alpha-1 activity refers to a
polypeptide comprising thymosin-alpha-1 or a variant or homolog
thereof. Thymosin-alpha-1 (TA1) is a 28-amino acid peptide and
includes synthetic forms of a naturally occurring hormone that
circulates in the thymus. TA1 stimulate thymocyte growth and
differentiation, production of IL-2, T cell IL-2 receptors,
IFN-.gamma. and IFN-.alpha.. Dosing regimes for TA1 are well known.
In any case doses in humans can be over a wide range such as 1 to
100 mg/dose.
[0087] The disclosure thus provides administering alpha thymosin
peptides ("thymosin peptides") to enhance cancer therapy with a
replication competent retroviral vector of the disclosure
comprising heterologous gene encoding a polypeptide having cytosine
deaminase activity. Thymosin peptides include thymosin alpha 1
("TA1"), and peptides having structural homology to TA1. TA1 is a
peptide having the amino acid sequence
Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-11e-Thr-Thr-Lys-Asp-Leu-Lys-Glu-L-
ys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn (SEQ ID NO:73) The amino
acid sequence of TA1 is disclosed in U.S. Pat. No. 4,079,137, the
disclosure of which is hereby incorporated by reference. TA1 is a
non-glycosylated 28-amino acid peptide having an acetylated
N-terminus, and a molecular weight of about 3108. A synthetic
version of TA1 is commercially available in certain countries under
the trade name ZADAXIN.RTM..
[0088] It is believed that thymosin peptides (e.g., TA1), among
other things, activate Toll-like Receptor 9 (TLR), resulting in
increases in Th1 cells, B cells, and NK cells, thereby leading to
enhancement of vaccine effectiveness. For example, TA1 may increase
or enhance lymphocytic infiltration, secretion of chemotactic
cytokines, maturation and differentiation of dendritic cells,
secretion of thymopoeitic cytokines including IFN-alpha, IL-7, and
IL-15, and B cell production of antibodies.
[0089] The thymosin peptides that find use with the vectors and
methods of the disclosure include naturally occurring TA1 (e.g.,
TA1 purified or isolated from tissues), as well as synthetic TA1
and recombinant TA1. In some embodiments, the thymosin peptide
comprises the amino acid sequence of SEQ ID NO:73 (where an
acylated, e.g., acetylated, N-terminus is optional). In some
embodiments, the thymosin peptide comprises an amino acid sequence
that is substantially similar to TA1, and maintains the
immunomodulatory activity of TA1. The substantially similar
sequence may have, for example, from about 1 to about 10 amino acid
deletions, insertions, and/or substitutions (collectively) with
respect to TA1. For example, the thymosin peptide may have from
about 1 to about 5 (e.g., 1, 2, or 3) amino acid insertions,
deletions, and/or substitutions (collectively) with respect to TA1
so long as the peptide has one or more activities associated with a
naturally occurring thymosin.
[0090] Thus, a thymosin peptide useful in the methods of the
disclosure may comprise an abbreviated TA1 sequence, for example,
having deletions of from 1 to about 10 amino acids, or from about 1
to 5 amino acids, or 1, 2 or 3 amino acids with respect to TA1.
Such deletions may be at the N- or C-terminus, and/or internal, so
long as the activity of the peptide is substantially maintained.
Alternatively, or in addition, the substantially similar sequence
may have from about 1 to about 5 amino acid insertions (e.g., 1, 2,
or 3 amino acid insertions) with respect to TA1, where the
immunomodulatory activity of TA1 is substantially maintained.
Alternatively, or in addition, the substantially similar sequence
may have from 1 to about 10 amino acid substitutions, where the
immunomodulatory activity is substantially maintained. For example,
the substantially similar sequence may have from 1 to about 5, or
1, 2, or 3 amino acid substitutions, which may include conservative
and non-conservative substitutions. In some embodiments, the
substitutions are conservative. Generally, conservative
substitutions include substitutions of a chemically similar amino
acid (e.g., polar, non-polar, or charged). Substituted amino acids
may be selected from the standard 20 amino acids or may be a
non-standard amino acid (e.g., a conserved non-standard amino
acid).
[0091] In some embodiments, the thymosin peptide comprises an amino
acid sequence having at least 70% sequence identity to SEQ ID
NO:73, while maintaining the activity of a naturally occurring TA1.
For example, the thymosin peptide may comprise an amino acid
sequence having at least 80%, 90%, or 95% sequence identity to SEQ
ID NO:73. The thymosin peptide may comprise an amino acid sequence
having 100% sequence identity to SEQ ID NO:73. In all cases, the
N-terminus may be optionally acylated (e.g., acetylated) or
alkylated, for example, with a C1-10 or C1-C7 acyl or alkyl
group.
[0092] The disclosure provides methods for treating cell
proliferative disorders such as cancer and neoplasms comprising
administering an RCR vector of the disclosure prior to,
simultaneously with or following administration of a thymosin
peptide. In another embodiment the combination of RCR and thymosin
may also be followed by treatment with a chemotherapeutic agent or
anti-cancer agent. In one aspect, the RCR vector is administered to
a subject for a period of time prior to administration of the
chemotherapeutic or anti-cancer agent that allows the RCR to infect
and replicate. The subject is then treated with a chemotherapeutic
agent or anti-cancer agent for a period of time and dosage to
reduce proliferation or kill the cancer cells. In one aspect, if
the treatment with the chemotherapeutic or anti-cancer agent
reduces, but does not kill the cancer/tumor (e.g., partial
remission or temporary remission), the subject may then be treated
with a non-toxic therapeutic agent (e.g., 5-FC) that is converted
to a toxic therapeutic agent in cells expression a cytotoxic gene
(e.g., cytosine deaminase) from the RCR. The methods and
compositions of the disclosure are useful in other combination
therapies, for example, therapies with Thymosin alpha-1
(Zadaxin.TM.), trastuzumab (Herceptin), Leucovorin and other folic
acid analogues, or other promoters of 5-FU activity (D. Papamichael
Stem Cells 18:166-175 2000) such as dihydropyrimidine dehydrogenase
[DPD] inhibitors [e.g. 5-Chloro-2,4-Dihydroxypyridine--Cdhp]) whose
action is targeted, rather than systemic, when used in conjunction
with the tumor targeted 5-FU production from 5-FC administration
and CD expression from the vector of disclosure.
[0093] Leucovorin or other folic acid analogues promote 5-FU
binding to thymidilate synthase, thereby inactivating this key
enzyme in nucleic acid biosynthesis, and enhancing the efficacy of
5-FU.
[0094] DPD inhibitors block the activity of dihydropyrimdine
dehydrogenasean enzyme that normally degrades about 80% of
systemically administered 5-FU. DPD inhibition results in increased
retention of 5-FU and frequently make 5-FU very much more toxic. In
fact this can be life threatening in patients that have DPD
deficiency (Ezeldin & Diasio Clinical Colorectal Cancer, Vol.
4, No. 3, 181-189, 2004). However, in the vectors of the
disclosure, 5-FU is only produced locally in the tumor, and hence
the increased toxicity is confined to the area of the tumor, where
it is a benefit.
[0095] The disclosure provides a method of treating a subject
having a cell proliferative disorder. The subject can be any
mammal, and is preferably a human. The subject is contacted with a
recombinant replication competent retroviral vector of the
disclosure. The contacting can be in vivo or ex vivo. Methods of
administering the retroviral vector of the disclosure are known in
the art and include, for example, systemic administration, topical
administration, intraperitoneal administration, intra-muscular
administration, intracranial, cerebrospinal, as well as
administration directly at the site of a tumor or
cell-proliferative disorder. Other routes of administration are
known in the art.
[0096] Thus, the disclosure includes various pharmaceutical
compositions useful for treating a cell proliferative disorder. The
pharmaceutical compositions according to the disclosure are
prepared by bringing a retroviral vector containing a heterologous
polynucleotide sequence useful in treating or modulating a cell
proliferative disorder according to the disclosure into a form
suitable for administration to a subject using carriers, excipients
and additives or auxiliaries. Frequently used carriers or
auxiliaries include magnesium carbonate, titanium dioxide, lactose,
mannitol and other sugars, talc, milk protein, gelatin, starch,
vitamins, cellulose and its derivatives, animal and vegetable oils,
polyethylene glycols and solvents, such as sterile water, alcohols,
glycerol and polyhydric alcohols. Intravenous vehicles include
fluid and nutrient replenishers. Preservatives include
antimicrobial, anti-oxidants, chelating agents and inert gases.
Other pharmaceutically acceptable carriers include aqueous
solutions, non-toxic excipients, including salts, preservatives,
buffers and the like, as described, for instance, in Remington's
Pharmaceutical Sciences, 15th ed. Easton: Mack Publishing Co.,
1405-1412, 1461-1487 (1975) and The National Formulary XIV., 14th
ed. Washington: American Pharmaceutical Association (1975), the
contents of which are hereby incorporated by reference. The pH and
exact concentration of the various components of the pharmaceutical
composition are adjusted according to routine skills in the art.
See Goodman and Gilman's The Pharmacological Basis for Therapeutics
(7th ed.).
[0097] For example, and not by way of limitation, a retroviral
vector useful in treating a cell proliferative disorder will
include an amphotropic ENV protein, GAG, and POL proteins, a
promoter sequence in the U3 region retroviral genome, and all
cis-acting sequence necessary for replication, packaging and
integration of the retroviral genome into the target cell.
[0098] The following Examples are intended to illustrate, but not
to limit the disclosure. While such Examples are typical of those
that might be used, other procedures known to those skilled in the
art may alternatively be utilized.
EXAMPLES
Example 1
Modification of Vector Backbone of pACE-GFPemd to pAC3-GFPemd and
Insertion of Cytosine Deaminase Gene Sequences in Place of GFP
[0099] The previous back bone of the pACE-GFPemd plasmid (U.S. Pat.
No. 6,899,871, Wang et al. Hum Gene Ther 14:117 2003) was modified
in 3 ways as shown in FIG. 3E. The modifications were made by
PCR-mediated, oligonucleotide-directed mutagenesis (Logg et al., J.
Mol Biol 369: 1214, 2007; see also "Molecular Biology and
Biotechnology" Eds. J M. Walker, R. Rapley, Royal Society of
Chemistry, London UK, 2000). The following modifications were made.
1) The nucleic acid sequence at the p15 region at the 3' end of the
amphotropic env gene was originally derived from the ecotropic
envelope--this sequence was replaced by the corresponding sequence
from the 4070A amphotropic envelope; the encoded envelope amino
acids are identical in the two constructs. 2) The IRES sequence 3'
end was modified to allow easier insertion of transgenes of choice
with insertion of a PstI1 site and small imperfect repeats at
either end of the IRES transgene site were removed. 3) Residual
viral sequence downstream of the 3'LTR was removed. The resultant
plasmid is pACE-emdGFP (aka pACE-GFP, pACE-eGFP and T5.0006) was
used as a basis for the vectors encoding cytosine deaminase and
variants. Two methods of inserting the coding sequence cassettes
were used initially. The first method resulted in the sequence
5'TTATAAT3' (SEQ ID NO:74), and the second in the sequence
5'TTATAA3'(SEQ ID NO:75) immediately upstream of the ATG start
codon. The second method was simpler, as it involved simple PstI1
and Not1 enzyme cuts in the vector and the synthetic cytosine
deaminase genes, followed by religation. Vectors with cytosine
deaminase inserts were made both ways with the CDopt (CD1) and the
CDopt+3pt (CD2) (see FIG. 2) coding sequences and infectious virus
preps made by transient transfection of 293T cells as described in
Example 3. U87 cells were then infected in culture, at an MOI of
0.1, and the cells grown until 100% infected. Cell extracts of 100%
infected cells were assayed for cytosine deaminase activity as
described in Example 6 and the specific activity of the enzyme was
found to be equivalent for constructs with either upstream
sequence, that were otherwise identical. Therefore in the table in
FIG. 2, pACE-eGFP (T5.0006) and pACE-yCD (T5.0007) have the first
upstream sequence, while all other constructs that were further
tested have the second. Subsequently vectors with different gene
inserts have been routinely constructed with straightforward PStI1
and Not I cuts.
[0100] See FIG. 3A below for a diagram of the vector construct for
the initial transfected replication-competent retrovirus. CMV is
the human CMV immediate early promoter, U3, R and U5 are the
corresponding regions of the viral long terminal repeat (LTR). Gag,
pol and env are the viral protein coding regions. FIGS. 3B and 3D
shows the plasmid structure and a sequence of the disclosure.
[0101] The vector of the disclosure provides a number of
differences compared to the vector of Tai et al., Mol. Ther.
12:842, 2005. The Tai et al. vector has been altered to eliminate
about 70 bp of MLV sequence downstream from the 3'LTR. The DNA
sequence downstream of the ClaI site in the envelope was changed to
an amphotropic envelope sequence. This change does not change the
amino acid sequence of the envelope. In addition, small repeats on
either side of the IRES-CD cassette have been eliminated to avoid
instability due to homologous recombination. These changes also
unexpectedly provided increased stability of the vector during
replication and passaging in host cells (FIG. 5).
[0102] It is recognized that after reverse transcription and the
first integration event into treated cells, the DNA provirus and
any subsequent progeny retrovirus has a conventional LTR structure
from MLV on either end. This configuration has been shown to be
stable after multiple cycles of infection (See FIG. 5 below).
Example 2
Genetic Enhancements to the Wild Type Yeast Cytosine Deaminase
Gene
[0103] Two sets of changes have been made: (1) three positional
mutations which change three amino acids (A23L, I140L and V108I) to
increase thermal stability of the yeast cytosine deaminase protein
and (2) additional gene sequence modifications to enhance human
codon usage sequences to improve protein translation efficiency in
human cells without further changes to the amino acid sequence.
[0104] Sequence design for CD included CD-optimized, CD-UPRT (+/-
linker) and CD-OPRTase (+/- linker). The final cytosine deaminase
coding sequence can comprise at the 5' end a PSI1 site (full
length) and 3' end NotI site plus poly A tail for PSI1/Not1
cassette based strategy. Sequences cassettes were ordered from, and
provided by, a commercial vendor (BioBasic Inc., Ontario,
Canada).
[0105] The following sequence comprising a yeast cytosine deaminase
was used for cloning, optimizing and mutation (the boxed nucleic
acids comprise the restriction sites--PsiI and NotI--used in
subsequent methods for cloning:
TABLE-US-00002 (SEQ ID NO: 43) ##STR00001##
TATGAGGAGGCGGCCTTAGGTTACAAAGAGGGTGGTGTTCCTATTGGCGGATGTCTTATCAATAACA
AAGACGGAAGTGTTCTCGGTCGTGGTCACAACATGAGATTTCAAAAGGGATCCGCCACACTACATGG
TGAGATCTCCACTTTGGAAAACTGTGGGAGATTAGAGGGCAAAGTGTACAAAGATACCACTTTGTAT
ACGACGCTGTCTCCATGCGACATGTGTACAGGTGCCATCATCATGTATGGTATTCCACGCTGTGTTG
TCGGTGAGAACGTTAATTTCAAAAGTAAGGGCGAGAAATATTTACAAACTAGAGGTCACGAGGTTGT
TGTTGTTGACGATGAGAGGTGTAAAAAGATCATGAAACAATTTATCGATGAAAGACCTCAGGATTGG
##STR00002## AAAAGGGGGG
The following Table summarizes the genes and resulting plasmid
vectors that were made and their names.
TABLE-US-00003 TABLE Vector constructs and names Identity Reference
Original 5'LTR Trans- Code name Name Prom Envelope Vector IRES gene
3'LTR T5.0000 pACE-yCD pACE-CD CMV Ampho pACE EMCV Wt yeast MLV U3
(Tai et al. (4070A) CD 2005) T5.0001 pAC3-yCD1 CDopt CMV Ampho pAC3
EMCV modified MLV U3 sequence (4070A) CD T5.0002 pAC3-yCD2
CDopt+3pt CMV Ampho pAC3 EMCV Modified MLV U3 (4070A) CD T5.0003
pAC3-yCD2-U Cdopt+3pt- CMV Ampho pAC3 EMCV CD2- MLV U3 UPRT (4070A)
UPRT T5.0004 pAC3-yCD2-O CDopt+3pt- CMV Ampho pAC3 EMCV CD2- MLV U3
OPRT (4070A) OPRT T5.0005 pAC3-yCD2-LO CDopt+3pt- CMV Ampho pAC3
EMCV CD2-L- MLV U3 LINK-OPRT (4070A) OPRT T5.0006 pAC3-eGFP
pAC3-emd, CMV Ampho pAC3 EMCV Emerald MLV U3 pAC3GFP (4070A) GFP
T5.0007 pAC3-yCD pAC3-yCD CMV Ampho pAC3 EMCV Wt yeast MLV U3
(4070A) CD
[0106] The replication competent retroviral vector described by
Kasahara et al. pACE-CD (U.S. Pat. No. 6,899,871, the disclosure of
which is incorporated herein) was used as a basis for additional
modifications. A vector (pAC3-yCD) was modified to express a
modified yeast cytosine deaminase gene as described herein and was
used in the constructs. See FIG. 3A below for a diagram of the
vector construct for the initial transfected replication-competent
retrovirus. CMV is the human CMV immediate early promoter, U3, R
and U5 are the corresponding regions of the viral long terminal
repeat (LTR). Gag, pol and env are the viral protein coding
regions. FIGS. 3B and 3D shows the plasmid structure and a sequence
of the disclosure.
[0107] After the genes were synthesized at a contractor (Bio Basic
Inc., Markham, Ontario, Canada) they were inserted into the
Psi1-Not1 site of the pAC3 vector backbone (FIG. 3). The plasmid
backbone was normally generated by cutting the plasmid pAC3-eGFP
with PsiI and NotI and purifying the large (about 11 kb) fragment
encoding the plasmid and retroviral backbone)
[0108] A. Humanized Codon Optimized CD Gene (CD-Opt, Aka CD1,
T5.0001).
[0109] A comparison of a human codon optimized cytosine deaminase
of Conrad et al. and PCT WO 99/60008 indicates 91 total codons
optimized in both, 36 codons identical, 47 codons had third base
pair changes (all encode same amino acid) and 9 codons were
different (however they encoded same amino acid). Of the 9 codons
that differed:
TABLE-US-00004 AGC (Ser) to TCC (Ser) CGT (Arg) to AGG (Arg) CCA
(Pro) to CCT (Pro)
[0110] All have equivalent GC content and encode the same amino
acid. The native yeast gene sequence above was separately codon
optimized to give the following CD gene (CD1) and was called
T5.0001 when inserted into the plasmid vector pAC3 which encodes
the replication competent retrovirus (RCR) with IRES.
TABLE-US-00005 (SEQ ID NO: 44) ##STR00003##
AGGCCGCCCTGGGCTACAAGGAGGGCGGCGTGCCTATCGGCGGCTGTCTGATCAACAACAAGGACGG
CAGTGTGCTGGGCAGGGGCCACAACATGAGGTTCCAGAAGGGCTCCGCCACCCTGCACGGCGAGATC
TCCACCCTGGAGAACTGTGGCAGGCTGGAGGGCAAGGTGTACAAGGACACCACCCTGTACACCACCC
TGTCCCCTTGTGACATGTGTACCGGCGCTATCATCATGTACGGCATCCCTAGGTGTGTGGTGGGCGA
GAACGTGAACTTCAAGTCCAAGGGCGAGAAGTACCTGCAAACCAGGGGCCACGAGGTGGTGGTTGTT
GACGATGAGAGGTGTAAGAAGATCATGAAGCAGTTCATCGACGAGAGGCCTCAGGACTGGTTCGAGG
##STR00004## G.
[0111] B. Heat Stabilized CD Gene.
[0112] Additional modifications were made to enhance the stability
of the cytosine deaminase. Genetic enhancements to the wild type
yeast cytosine deaminase gene were made to include three positional
mutations which change three amino acids (A23L, I140L and V108I) to
increase thermal stability of the yeast cytosine deaminase
protein.
[0113] The following primer pairs were used in the generation of
the gene for the cytosine deaminase polypeptide of the
disclosure:
TABLE-US-00006 Site directed mutagenesis primers: Primers sense:
(SEQ ID NO: 45) 5'-tcgaggatatcggcgagtgaaacccgttattctttttggc-3'
Primers antisense: (SEQ ID NO: 46)
5'-gccaaaaagaataacgggtttcactcgccgatatcctcga-3' Primers sense: (SEQ
ID NO: 47) 5'tcggcgagtgatccggcggcggcgcctccggcggcggcgcctccggcg
gcggcgcctccggcggcggcgccaacccgttatt-3' Primers antisense: (SEQ ID
NO: 48) 5'-aataacgggttggcgccgccgccggaggcgccgccgccggaggcgcc
gccgccggaggcgccgccgccggatcactcgccga-3'
[0114] To increase the stability of the native yeast CD protein,
three amino acid substitutions were engineered into the protein.
These substitutions were alone or in combination with human codon
optimization.
[0115] The three amino acid substitutions are: A23L, V108I, I140L.
A sequence encoding these substitutions is shown below.
TABLE-US-00007 (SEQ ID NO: 3)
ATGGTGACAGGGGGAATGGCAAGCAAGTGGGATCAGAAGGGTATGGACATTGCCTATGAGGAGGCGT
TATTAGGTTACAAAGAGGGTGGTGTTCCTATTGGCGGATGTCTTATCAATAACAAAGACGGAAGTGT
TCTCGGTCGTGGTCACAACATGAGATTTCAAAAGGGATCCGCCACACTACATGGTGAGATCTCCACT
TTGGAAAACTGTGGGAGATTAGAGGGCAAAGTGTACAAAGATACCACTTTGTATACGACGCTGTCTC
CATGCGACATGTGTACAGGTGCCATCATCATGTATGGTATTCCACGCTGTGTCATCGGTGAGAACGT
TAATTTCAAAAGTAAGGGCGAGAAATATTTACAAACTAGAGGTCACGAGGTTGTTGTTGTTGACGAT
GAGAGGTGTAAAAAGTTAATGAAACAATTTATCGATGAAAGACCTCAGGATTGGTTTGAAGATATTG
##STR00005##
[0116] The encoded polypeptide comprises the following sequence
(substituted amino acids in underlined):
TABLE-US-00008 (SEQ ID NO: 4) 1
MVTGGMASKWDQKGMDIAYEEALLGYKEGGVPIGGCLINNKDGSVLGRGHNMRFQKGSAT 61
LHGEISTLENCGRLEGKVYKDTTLYTTLSPCDMCTGAIIMYGIPRCVIGENVNFKSKGEK 121
YLQTRGHEVVVVDDERCKKLMKQFIDERPQDWFEDIGE-
[0117] Final construct design that integrates 3 amino acid
substitutions A23L/V108I/I140L utilizing preferred codons and uses
preferred human codon usage for entire sequence (this gene is
called CDopt+3pt [aka CD2] and T5.0002 when inserted into the
plasmid vector pAC3 which encodes the RCR with IRES.
TABLE-US-00009 (SEQ ID NO: 49) 1
ATGGTGACCGGCGGCATGGCCTCCAAGTGGGATCAAAAGGGCATGGATATCGCTTACGAG 61
GAGGCCCTGCTGGGCTACAAGGAGGGCGGCGTGCCTATCGGCGGCTGTCTGATCAACAAC 121
AAGGACGGCAGTGTGCTGGGCAGGGGCCACAACATGAGGTTCCAGAAGGGCTCCGCCACC 181
CTGCACGGCGAGATCTCCACCCTGGAGAACTGTGGCAGGCTGGAGGGCAAGGTGTACAAG 241
GACACCACCCTGTACACCACCCTGTCCCCTTGTGACATGTGTACCGGCGCTATCATCATG 301
TACGGCATCCCTAGGTGTGTGATCGGCGAGAACGTGAACTTCAAGTCCAAGGGCGAGAAG 361
TACCTGCAAACCAGGGGCCACGAGGTGGTGGTTGTTGACGATGAGAGGTGTAAGAAGCTG 421
ATGAAGCAGTTCATCGACGAGAGGCCTCAGGACTGGTTCGAGGATATCGGCGAGTGATAA
Underlined codons denote preferred codons for amino acid
substitutions.
[0118] Protein translation sequence alignment indicates preferred
codon changes and amino acid substitutions result in desired
protein structure:
[0119] CD-optimized sequence design (human codon preference+3 amino
acid substitutions)
TABLE-US-00010 (SEQ ID NO: 50) ##STR00006##
TACGAGGAGGCCCTGCTGGGCTACAAGGAGGGCGGCGTGCCTATCGGCGGCTGTCTGATCAACAACA
AGGACGGCAGTGTGCTGGGCAGGGGCCACAACATGAGGTTCCAGAAGGGCTCCGCCACCCTGCACGG
CGAGATCTCCACCCTGGAGAACTGTGGCAGGCTGGAGGGCAAGGTGTACAAGGACACCACCCTGTAC
ACCACCCTGTCCCCTTGTGACATGTGTACCGGCGCTATCATCATGTACGGCATCCCTAGGTGTGTGA
TCGGCGAGAACGTGAACTTCAAGTCCAAGGGCGAGAAGTACCTGCAAACCAGGGGCCACGAGGTGGT
GGTTGTTGACGATGAGAGGTGTAAGAAGCTGATGAAGCAGTTCATCGACGAGAGGCCTCAGGACTGG
##STR00007## AAAAGGGGGG
[0120] C. Construction of CD-UPRT Fusion Gene (CDopt+3pt-UPRT, [Aka
CDopt-UPRT and CD2-UPRT], T5.0003 in the pAC3 Plasmid RCR
Vector).
[0121] A fusion construct was also developed comprising a CD
polypeptide as described above linked to a UPRT polypeptide to
generate a CD-optimized-UPRT. The following primers were used to
delete the stop-start between the CD and UPRT.
Primer Sequences:
TABLE-US-00011 [0122] Primer Name Primer Sequence (5' to 3') (SEQ
ID NO:) del118-123 5'-tcgaggatatcggcgagtgaaacccgttattctttttggc-3'
(51) del118-123-antisense
5'-gccaaaaagaataacgggtttcactcgccgatatcctcga-3' (52) Energy Cost
Length Duplex Energy of Primer Name (nt.) Tm at 68.degree. C.
Mismatches del118-123 40 79.06.degree. C. -44.37 kcal/mole 21.1%
del118-123-antisense 40 79.06.degree. C. -47.95 kcal/mole 20.3%
Primer Name Primer-Template Duplex del118-123 (SEQ ID NOs: 51 and
53, respectively ##STR00008## del118-123-anti-sensense (SEQ ID NO:
54 and 52 respectively) ##STR00009##
[0123] The resulting fusion polynucleotide comprises 1296 bp and
the sequence set forth immediately below:
TABLE-US-00012 (SEQ ID NO: 55) ##STR00010##
TACGAGGAGGCCCTGCTGGGCTACAAGGAGGGCGGCGTGCCTATCGGCGGCTGTCTGATCAACAACA
AGGACGGCAGTGTGCTGGGCAGGGGCCACAACATGAGGTTCCAGAAGGGCTCCGCCACCCTGCACGG
CGAGATCTCCACCCTGGAGAACTGTGGCAGGCTGGAGGGCAAGGTGTACAAGGACACCACCCTGTAC
ACCACCCTGTCCCCTTGTGACATGTGTACCGGCGCTATCATCATGTACGGCATCCCTAGGTGTGTGA
TCGGCGAGAACGTGAACTTCAAGTCCAAGGGCGAGAAGTACCTGCAAACCAGGGGCCACGAGGTGGT
GGTTGTTGACGATGAGAGGTGTAAGAAGCTGATGAAGCAGTTCATCGACGAGAGGCCTCAGGACTGG
TTCGAGGATATCGGCGAGAACCCGTTATTCTTTTTGGCTTCTCCATTCTTGTACCTTACATATCTTA
TATATTATCCAAACAAAGGGTCTTTCGTTAGCAAACCTAGAAATCTGCAAAAAATGTCTTCGGAACC
ATTTAAGAACGTCTACTTGCTACCTCAAACAAACCAATTGCTGGGTTTGTACACCATCATCAGAAAT
AAGAATACAACTAGACCTGATTTCATTTTCTACTCCGATAGAATCATCAGATTGTTGGTTGAAGAAG
GTTTGAACCATCTACCTGTGCAAAAGCAAATTGTGGAAACTGACACCAACGAAAACTTCGAAGGTGT
CTCATTCATGGGTAAAATCTGTGGTGTTTCCATTGTCAGAGCTGGTGAATCGATGGAGCAAGGATTA
AGAGACTGTTGTAGGTCTGTGCGTATCGGTAAAATTTTAATTCAAAGGGACGAGGAGACTGCTTTAC
CAAAGTTATTCTACGAAAAATTACCAGAGGATATATCTGAAAGGTATGTCTTCCTATTAGACCCAAT
GCTGGCCACCGGTGGTAGTGCTATCATGGCTACAGAAGTCTTGATTAAGAGAGGTGTTAAGCCAGAG
AGAATTTACTTCTTAAACCTAATCTGTAGTAAGGAAGGGATTGAAAAATACCATGCCGCCTTCCCAG
AGGTCAGAATTGTTACTGGTGCCCTCGACAGAGGTCTAGATGAAAACAAGTATCTAGTTCCAGGGTT
##STR00011## TTTAGTCTCCAGAAAAAGGGGGG
[0124] D. Construction of CD-Linker UPRT Fusion Gene
(CDopt+3pt-LINK-UPRT [Aka CDopt-LINKER-UPRT and CD2-L-UPRT].
[0125] A fusion construct was also developed by cloning a linker
(Ser-Gly-Gly-Gly-Gly).sub.4 (SEQ ID NO:56) domain between and in
frame with the CD polypeptide and the UPRT polypeptide to generated
a CD-optimized-linker-UPRT sequence. The following primers were
used to insert the linker.
TABLE-US-00013 Primer Name Primer Sequence (5' to 3')(SEQ ID NO:)
ins_60nt_after_477 5'-
tcggcgagtgatccggcggcggcgcctccggcggcggcgcctccggcg
gcggcgcctccggcggcggcgccaacccgttatt-3'(57) ins_60nt_after_477- 5'-
antisense aataacgggttggcgccgccgccggaggcgccgccgccggaggcgcc
gccgccggaggcgccgccgccggatcactcgccga-3'(58) Energy Cost Length
Duplex Energy at of Primer Name (nt.) Tm 68.degree. C. Mismatches
ins_60nt_after_477 82 79.77.degree. C. -30.19 kcal/mole 83.3%
ins_60nt_after_477- 82 79.77.degree. C. -32.31 kcal/mole 82.2%
antisense
[0126] The resulting construct has size: 1356 bp and the sequence
immediately below:
TABLE-US-00014 (SEQ ID NO: 59) ##STR00012##
TACGAGGAGGCCCTGCTGGGCTACAAGGAGGGCGGCGTGCCTATCGGCGGCTGTCTGATCAACAACA
AGGACGGCAGTGTGCTGGGCAGGGGCCACAACATGAGGTTCCAGAAGGGCTCCGCCACCCTGCACGG
CGAGATCTCCACCCTGGAGAACTGTGGCAGGCTGGAGGGCAAGGTGTACAAGGACACCACCCTGTAC
ACCACCCTGTCCCCTTGTGACATGTGTACCGGCGCTATCATCATGTACGGCATCCCTAGGTGTGTGA
TCGGCGAGAACGTGAACTTCAAGTCCAAGGGCGAGAAGTACCTGCAAACCAGGGGCCACGAGGTGGT
GGTTGTTGACGATGAGAGGTGTAAGAAGCTGATGAAGCAGTTCATCGACGAGAGGCCTCAGGACTGG
TTCGAGGATATCGGCGAGTCCGGCGGCGGCGCCTCCGGCGGCGGCGCCTCCGGCGGCGGCGCCTCCG
GCGGCGGCGCCAACCCGTTATTCTTTTTGGCTTCTCCATTCTTGTACCTTACATATCTTATATATTA
TCCAAACAAAGGGTCTTTCGTTAGCAAACCTAGAAATCTGCAAAAAATGTCTTCGGAACCATTTAAG
AACGTCTACTTGCTACCTCAAACAAACCAATTGCTGGGTTTGTACACCATCATCAGAAATAAGAATA
CAACTAGACCTGATTTCATTTTCTACTCCGATAGAATCATCAGATTGTTGGTTGAAGAAGGTTTGAA
CCATCTACCTGTGCAAAAGCAAATTGTGGAAACTGACACCAACGAAAACTTCGAAGGTGTCTCATTC
ATGGGTAAAATCTGTGGTGTTTCCATTGTCAGAGCTGGTGAATCGATGGAGCAAGGATTAAGAGACT
GTTGTAGGTCTGTGCGTATCGGTAAAATTTTAATTCAAAGGGACGAGGAGACTGCTTTACCAAAGTT
ATTCTACGAAAAATTACCAGAGGATATATCTGAAAGGTATGTCTTCCTATTAGACCCAATGCTGGCC
ACCGGTGGTAGTGCTATCATGGCTACAGAAGTCTTGATTAAGAGAGGTGTTAAGCCAGAGAGAATTT
ACTTCTTAAACCTAATCTGTAGTAAGGAAGGGATTGAAAAATACCATGCCGCCTTCCCAGAGGTCAG
AATTGTTACTGGTGCCCTCGACAGAGGTCTAGATGAAAACAAGTATCTAGTTCCAGGGTTGGGTGAC
##STR00013## TCCAGAAAAAGGGGGG
[0127] E. Construction of CD-OPRT Fusion Gene (CDopt+3pt-OPRT [Aka
CDopt-OPRT and CD2-OPRT], T5.0004 when Inserted into the pAC3
Plasmid RCR Vector).
[0128] A fusion construct was also developed comprising a CD
polypeptide as described above linked to an OPRT polypeptide to
generate a CD-optimized-OPRTase (CD humanized+3ptmutation+OPRTase
functional domain human).
[0129] The resulting construct comprises a size of 1269 bp and the
sequence immediately below:
TABLE-US-00015 (SEQ ID NO: 60) ##STR00014##
TACGAGGAGGCCCTGCTGGGCTACAAGGAGGGCGGCGTGCCTATCGGCGGCTGTCTGATCAACAACA
AGGACGGCAGTGTGCTGGGCAGGGGCCACAACATGAGGTTCCAGAAGGGCTCCGCCACCCTGCACGG
CGAGATCTCCACCCTGGAGAACTGTGGCAGGCTGGAGGGCAAGGTGTACAAGGACACCACCCTGTAC
ACCACCCTGTCCCCTTGTGACATGTGTACCGGCGCTATCATCATGTACGGCATCCCTAGGTGTGTGA
TCGGCGAGAACGTGAACTTCAAGTCCAAGGGCGAGAAGTACCTGCAAACCAGGGGCCACGAGGTGGT
GGTTGTTGACGATGAGAGGTGTAAGAAGCTGATGAAGCAGTTCATCGACGAGAGGCCTCAGGACTGG
TTCGAGGATATCGGCGAGGCGGTCGCTCGTGcagctttggggccattggtgacgggtctgtacgacg
tgcaggctttcaagtttggggacttcgtgctgaagagcgggctttcctcccccatctacatcgatct
gcggggcatcgtgtctcgaccgcgtcttctgagtcaggttgcagatattttattccaaactgcccaa
aatgcaggcatcagttttgacaccgtgtgtggagtgccttatacagctttgccattggctacagtta
tctgttcaaccaatcaaattccaatgcttattagaaggaaagaaacaaaggattatggaactaagcg
tcttgtagaaggaactattaatccaggagaaacctgtttaatcattgaagatgttgtcaccagtgga
tctagtgttttggaaactgttgaggttcttcagaaggagggcttgaaggtcactgatgccatagtgc
tgttggacagagagcagggaggcaaggacaagttgcaggcgcacgggatccgcctccactcagtgtg
tacattgtccaaaatgctggagattctcgagcagcagaaaaaagttgatgctgagacagttgggaga
gtgaagaggtttattcaggagaatgtctttgtggcagcgaatcataatggttctcccctttctataa
aggaagcacccaaagaactcaGCTTCGGTGCACGTGCAGAGCTGCCCAGGATCCACCCAGTTGCATC
##STR00015##
[0130] F. Construction of CD-Linker-OPRT Fusion Gene
(CDopt+3pt-LINK-OPRT, [Aka CDopt-LINKER-OPRT and CD2-L-OPRT],
T5.0005 in the pAC3 plasmid RCR vector).
[0131] A fusion construct was also developed by cloning a linker
(Ser-Gly-Gly-Gly-Gly).sub.4) (SEQ ID NO:56) domain between and in
frame with the CD polypeptide and the OPRT polypeptide to generated
a CD-optimized-linker-OPRT sequence.
[0132] The resulting construct comprises a size of 1329 bp and the
sequence immediately below:
TABLE-US-00016 (SEQ ID NO: 61) ##STR00016##
TACGAGGAGGCCCTGCTGGGCTACAAGGAGGGCGGCGTGCCTATCGGCGGCTGTCTGATCAACAACA
AGGACGGCAGTGTGCTGGGCAGGGGCCACAACATGAGGTTCCAGAAGGGCTCCGCCACCCTGCACGG
CGAGATCTCCACCCTGGAGAACTGTGGCAGGCTGGAGGGCAAGGTGTACAAGGACACCACCCTGTAC
ACCACCCTGTCCCCTTGTGACATGTGTACCGGCGCTATCATCATGTACGGCATCCCTAGGTGTGTGA
TCGGCGAGAACGTGAACTTCAAGTCCAAGGGCGAGAAGTACCTGCAAACCAGGGGCCACGAGGTGGT
GGTTGTTGACGATGAGAGGTGTAAGAAGCTGATGAAGCAGTTCATCGACGAGAGGCCTCAGGACTGG
TTCGAGGATATCGGCGAGTCCGGCGGCGGCGCCTCCGGCGGCGGCGCCTCCGGCGGCGGCGCCTCCG
GCGGCGGCGCCGCGGTCGCTCGTGcagctttggggccattggtgacgggtctgtacgacgtgcaggc
tttcaagtttggggacttcgtgctgaagagcgggctttcctcccccatctacatcgatctgcggggc
atcgtgtctcgaccgcgtcttctgagtcaggttgcagatattttattccaaactgcccaaaatgcag
gcatcagttttgacaccgtgtgtggagtgccttatacagctttgccattggctacagttatctgttc
aaccaatcaaattccaatgcttattagaaggaaagaaacaaaggattatggaactaagcgtcttgta
gaaggaactattaatccaggagaaacctgtttaatcattgaagatgttgtcaccagtggatctagtg
ttttggaaactgttgaggttcttcagaaggagggcttgaaggtcactgatgccatagtgctgttgga
cagagagcagggaggcaaggacaagttgcaggcgcacgggatccgcctccactcagtgtgtacattg
tccaaaatgctggagattctcgagcagcagaaaaaagttgatgctgagacagttgggagagtgaaga
ggtttattcaggagaatgtctttgtggcagcgaatcataatggttctcccctttctataaaggaagc
acccaaagaactcaGCTTCGGTGCACGTGCAGAGCTGCCCAGGATCCACCCAGTTGCATCGAAGTAA
##STR00017##
[0133] FIG. 4 demonstrates that higher levels of the human codon
optimized with the three mutations for higher stability are
observed compared to wild type yCD protein in infected U-87
cells.
Example 3
Vector Production by Transient Transfection
[0134] Vector can be produced in a number of ways, but the first
step is to introduce the DNA vector into cells to allow production
of infectious particles, that can then be harvested from the cell
supernatant. Once infectious particles have been generated other
methods of production can be implemented by those skilled in the
art. Vector particles were generated by transient transfection of
293T cells (Pear et al. Proc Natl Acad Sci USA. 90:8392-8396
1993).
[0135] The 293T cells were thawed and put into culture, then
passaged twice in T-75 flasks containing 15 mL of the DMEM medium
that was prepared by mixing DMEM High Glucose medium
(Hyclone#30081, 500 mL) with FBS (Hyclone# SH30070, 50 mL),
L-Glutamine (Cellgro#25-005-CI, 5 mL), NEAA (Hyclone #SH30238, 5
mL), and Penicillin-strep (Cellgro#30-002-CI, 5 mL). The flasks
were incubated at 37.degree. C. and 5% CO.sub.2. After the 3.sup.rd
passage cells were seeded in 6 T-25's, each containing 5 mL of the
medium, at a cell density of 1.8.times.10.sup.6 cells/T-25 (or
7.2.times.10.sup.4 cells/cm.sup.2). One day after seeding the
T-25's, the cells were transfected with the T5.0002 plasmid that
expressed the viral vector using the Calcium Phosphate Transfection
Kit from Promega (Cat# E1200). Eighteen hours following
transfection, the media in one set of the flasks (3 flasks each
set) were replaced with fresh medium containing 10 mM NaB. The
media in the 2.sup.nd set of the flasks were not replaced, which
served as a control (zero NaB). Eight hours post NaB treatment the
media in all flasks were replaced with the fresh medium containing
no NaB. The expression was allowed to continue for both sets of
flasks until the next day (22 hours duration). The supernatants
from both sets of flasks were harvested and assayed for their
titers by qPCR expressed in Transducing Units (TU)/ml (see Example
4).
[0136] The titer results are shown in the following table.
TABLE-US-00017 Second titer (after storing Condition First titer at
-80.degree. C. for 68 days) Without NaB 1.5 (.+-.0.05) .times.
10.sup.6 TU/mL 1.2 (.+-.0.2) .times. 10.sup.6 TU/mL 10 mM NaB 1.4
(.+-.0.3) .times. 10.sup.6 TU/mL 7.0 (.+-.0.14) .times. 10.sup.5
TU/mL TU = transduction unit
[0137] Subsequent vector preparations were produced in this manner,
without sodium butyrate. Other vector plasmids (Table 2) have been
used in the same way to generate vector preparations with titers
between 1E5 TU/ml and 1E7 TU/ml. Such material can be further
purified and concentrated, if desired, as described below see also:
U.S. Pat. No. 5,792,643; T. Rodrigues et al. J Gene Med 9:233
2007.
[0138] In certain embodiments of the disclosure the dosing was
calculated by grams of brain weight. In such embodiments, the
dosing of a replication competent retroviral vector of the
disclosure useful in the methods for treatment can range from
10.sup.4 to 10.sup.6 TU per gram brain weight.
Example 4
Quantitative PCR Titering Assay
[0139] The functional vector concentration, or titer, is determined
using a quantitative PCR-based (qPCR) method. In this method,
vector is titered by infecting a transducible host cell line (e.g.
PC-3 human prostatic carcinoma cells, ATCC Cat# CRL-1435) with a
standard volume of vector and measuring the resulting amount of
provirus present within the host cells after transduction. The
cells and vector are incubated under standard culturing condition
(37.degree. C., 5% CO.sub.2) for 24 hr to allow for complete
infection prior to the addition of the anti-retroviral AZT to stop
vector replication. Next, the cells are harvested from the culture
dish and the genomic DNA (gDNA) is purified using an Invitrogen
Purelink gDNA purification kit and eluted from the purification
column with sterile RNase-/DNase-free water. The
A.sub.260/A.sub.280 absorbance ratio is measured on a
spectrophotometer to determine the concentration and relative
purity of the sample. The gDNA concentrations are normalized with
additional RNase-/DNase-free water to the lowest concentration of
any given set of gDNA preparations such that the input DNA for the
qPCR is constant for all samples analyzed. Genomic DNA purity is
further assessed by electrophoresis of an aliquot of each sample on
an ethidium bromide stained 0.8% agarose gel. If the sample passes
an A.sub.260/A.sub.280 absorbance range of 1.8-2.0 and shows a
single band of gDNA, then the sample is ready for qPCR analysis of
provirus copy number of the vector. Using primers that interrogate
the LTR region of the provirus (reverse-transcribed vector DNA and
vector DNA that is integrated into the host gDNA), qPCR is
performed to estimate the total number of transduction events that
occurred when the known volume of vector was used to transduce the
known number of cells. The number of transduction events per
reaction is calculated from a standard curve that utilizes a
target-carrying plasmid of known copy-number that is serial diluted
from 10.sup.7 to 10 copies and measured under identical qPCR
conditions as the samples. Knowing how many genomic equivalents
were used for each qPCR reaction (from the concentration previously
determined) and how many transduction events that occurred per
reaction, we determine the total number of transduction events that
occurred based on the total number of cells that were present at
the time of transduction. This value is the titer of the vector
after dilution into the medium containing the cells during the
initial transduction. To calculate the corrected titer value, the
dilution is corrected for by multiplying through by the volume of
culture and the volume of titer divided by the volume of titer.
These experiments are performed in replicate cultures and analyzed
by qPCR using triplicate measurements for each condition to
determine an average titer and with its associated standard
deviation and coefficient of variance.
Example 5
Vector Testing
[0140] In order to be effective vector constructs and their derived
infectious particles need to: (1) make good titer of virus by
transient transfection (see Examples 3 and 4); (2) be stable upon
multiple passages; (3) kill cells efficiently in the presence of
5-FC; and (4) express enzyme activity upon infection of target
cells. Example 3 shows that useful titers can be obtained from the
vectors.
[0141] Genetic Stability of Viral Vectors.
[0142] To demonstrate the stability the following experiment was
performed. Approximately 10.sup.6 naive U-87 cells were initially
infected with the viral vector at an MOI of 0.01, and grown until
fully infected to complete a single cycle of infection. Supernatant
is then repassed onto uninfected cells and the cycle repeated. In
this experiment, twelve cycles have been completed in duplicate
trials (FIG. 5 shows one of each of the duplicate trials; the other
duplicates gave similar results). Genomic stability of the yCD2 or
other transgene sequence is assessed by PCR amplification of the
integrated provirus from the infected cells using MLV specific
primers flanking the transgene insertion site. The appearance of
any bands smaller than full-length amplicon would be an indicator
of vector instability. FIG. 5 demonstrates that a vector of the
disclosure (T5.0007-comprising the modified vector and CD
heterologous polynucleotide) maintains stability for more passages
than pACE-CD. Furthermore T5.0003 is somewhat less stable while
T5.0004 and T5 appear about as stable as pACE-CD. pACE-CD has been
used in mouse tumor studies and shows good anti-tumor effects in
mouse models. However a more stable viral genome will be much more
potent and long lasting in treatment of animals and humans,
especially if multiple cycles of 5-FC treatment are required. Both
T5.0001 and T5.0002 are markedly more stable than even T5.0007, as
shown by the reduced presence of small bands at later passages
(FIG. 5), showing that silent changes in a protein coding sequence
or small changes that result in point mutations can lead to
unexpectedly superior properties with more stable vector
genomes.
[0143] Cell Killing Experiments.
[0144] The CellTiter 96 Aqueous One Solution Cell Proliferation
Assay (MTS) is a colorimetric method for determining the number of
viable cells in proliferation assays. We have utilized this assay
to determine cell growth kinetics, as well as to determine the dose
response of various cell lines to 5-Fluorocytosine (5-FC) and
5-Fluorouracil (5-FU).
[0145] Cells 100% infected with vector were seeded at 1000
cells/well in 96-well plates. They were monitored over an eight day
period following treatment with various concentrations of 5-FC
(5-FU for controls). An analysis of their cell growth was assessed
every two days utilizing Promega's Cell Titer 96 AQueous One
Solution reagent (MTS). Briefly, 20 .mu.l of MTS was mixed with 100
.mu.l media (as recommended by the manufacturer) and added to the
samples in the 96-well plate. The samples were incubated for 60
minutes in a 37.degree. C./5% CO.sub.2 incubator. Thereafter,
absorbance readings were taken on a plate reader at a 490 nm
wavelength.
[0146] FIG. 6A shows the results of an experiment that demonstrates
that the cytosine deaminase in cells expressing the yCD2 protein is
at least as active as that from cells expressing the wild type yCD
protein, by performing 5-FC titrations on U-87 cells infected
either with AC3-yCD2 (vector) or AC3-yCD (vector). Briefly, U-87
cells 5 days post infection at a multiplicity of infection of 0.1
(i.e. 100% infected) with either AC3-yCD (wild type CD) vector or
AC3-yCD2 (thermostabilized & codon optimized) vector were
subject to increasing amounts of 5-FC or 0.1 mM of 5-FU as a
positive control for 8 days. On day 8, cell cultures were assessed
for viability using an MTS assay (Promega CellTiter 96 AQUEOUS One
Solution Proliferation Assay). Data shows comparable killing
between the two retroviral vectors at increasing doses of 5-FC
treatment.
[0147] In similar in-vitro cell culture experiments with RG2 cells
(ATCC Cat# CRL-2433), the RG2 cell line was transduced with 5
different vectors (pACE-CD, T5.0001, T5.0002, T5.0004, and
T5.0007). It was subsequently subject to increasing concentrations
of 5-FC (5-FU for controls) for 8 days and monitored as described
above. The results are shown in FIG. 2. Concentrations of 0.01 mM
were sufficient to induce complete killing with all vectors tested
with the exception of wild type-yeast Cytosine Deaminase
(pACE-yCD). It was less sensitive and required 1.0 mM of 5-FC for
complete killing.
[0148] CD Expression Assay.
[0149] U87 cells were transduced at a multiplicity of infection
(MOI) of 0.1, cultivated for 5 days to allow viral spread and cells
from day 5 post transduction were harvested. The cells were then
collected by centrifugation at 800.times.g for 5 min. The
supernatant was aspirated away from the cell pellet and washed with
5 mL of phosphate buffered saline (PBS) and again centrifuged at
800.times.g for 5 min. The resulting cell pellet was taken up in
1.5 mL of PBS, resuspended by passage through a pipette tip and
placed in a freezer at -20.degree. C. Cells were lysed by a
freeze/thaw method. Previously resuspended cells were allowed to
thaw at room temperature, passed through a pipette tip, mixed with
protease inhibitor cocktail and again refrozen at -20.degree. C.
Previous to the enzyme assay, the sample was again thawed at room
temperature and passed through a pipette tip. The suspension was
then centrifuged at 14,000 rpm in a tabletop centrifuge for 5 min.
The supernatant was decanted away from the pellet and placed in a
fresh eppendorf tube and placed on ice.
[0150] yCD enzyme activity was assessed by using an HPLC assay. The
HPLC assay was performed on a Shimadzu LC20AT unit connected in
series with a photoarray detector and autoinjector. The solid phase
was a Hypersil BDS C.sub.18, HPLC column with a 5 .mu.m sphere size
and 4.0.times.250 mm column dimensions. The mobile phase was 50 mM
ammonium phosphate, pH 2.1, containing 0.01% tert-butylammonium
perchlorate and 5% methanol; the system was equilibrated at
22.degree. C. All reagents were ACS grade and solvents were HPLC
grade. A reaction mix was made consisting of 800 .mu.L with a final
concentration of 0.125 mg/mL 5-FC (1 mM) in PBS and placed in a 1.5
mL autosampler vial. The reaction was then initiated by adding 200
.mu.L of each cell lysate. The reaction/autosampler vials were
placed in the auto sampler and 5 .mu.L of the reaction mixture was
injected. Time points were taken periodically by retrieving a 5
.mu.L aliquot from each reaction vial and analyzing on the HPLC
column. The conversion rates of 5-FC to 5-FU were calculated by
comparing the peak areas with known amounts from a previously
generated standard curve of 5-FU. The rate of 5-FC conversion to
5-FU was derived by plotting the amount of 5-FU (in nmol) generated
against its corresponding time interval. Protein concentration for
the cell sample was derived and the Specific Activity of the cell
lysate samples were calculated by dividing the conversion rate
(nmol/min) by the amount of protein used in the assay in mg.
[0151] FIG. 6B shows the specific activity of various vectors after
5 days on transduction at an MOI of 0.1. The data demonstrate that
pACE-yCD (T5.0000)<pAC3-yCD1 (T5.0001)<pAC3-CD2 (T5.0002) in
terms of the specific activity of cytosine deaminase in tissue
culture cells.
Example 6
Vector Purification and Concentration
[0152] A vector of the disclosure is manufactured by transient
transfection on 293T cells (Example 3), followed by harvesting of
the cell supernatant, filtration, benzonase treatment,
diafiltration/concentration and dialysis. A further chromatography
column step may be included, known to those skilled in the art (see
for example U.S. Pat. No. 5,792,643; T. Rodriguez et al. J Gene Med
9:233 2007; WO2010148203. Vector is also produced from a
permanently infected cell line and processed as above (see for
example WO2010148203). Clinical material is released based on
standard testing such as sterility, mycoplasma and endotoxins, plus
product specific potency, strength, and identity testing. Titer is
determined as Transducing Units (TU) by PCR quantitation of
integrated viral vector DNA in target cells (Example 4). The final
product is targeted to have a titer of up to 10.sup.9 TU/ml
formulated in isotonic Tris-buffered sucrose solution, as a sterile
injectable.
[0153] In general, to accurately and precisely determine the
strength of vector lots, a quantitative PCR-based titer assay has
been developed (described in general terms in example 4). The
details of the assay procedure consist of the following steps:
[0154] Transduction.
[0155] Transductions are performed in a 12-well plate format using
the stable human prostate adenocarcinoma derived PC-3 cell line.
For each test sample, three dilutions of un-titered vector
preparation are used to transduce PC-3 cells in triplicate wells.
Viral replication is stopped 24 hours post-transduction with
azidothymidine (AZT). Cells are maintained for an additional 24-64
hours prior to harvesting and genomic DNA purification.
[0156] Genomic DNA Preparation.
[0157] Qiagen DNeasy DNA Minikits are used to prepare genomic DNA
from transduced harvested cells as per the manufacturer's protocol.
DNA concentrations and quality are assessed by direct absorbance
measurement using UV/vis spectrophotometry to determine the A260
and A260/A280 ratio.
[0158] Real-Time Quantitative PCR.
[0159] The BioRad CFX96 real-time PCR instrument or equivalent is
used for performing quantitative PCR. Provector copy numbers
present in each test sample are measured by using specific DNA
oligonucleotide primers in conjunction with a TaqMan probe designed
to amplify the integrated, or pro-retroviral, U3/Psi packaging
versus the CMV/Psi plasmid promoter. Vector titer is expressed
relative to a copy number standard curve. To generate the vector
copy number standard curve, genomic DNA from PC-3 cells is spiked
with a unique plasmid containing the pro-retroviral U3/Psi
sequence. Vector test sample titers are obtained by calculating the
number of transduced genomes in multiple dilutions using multiple
reactions per dilution.
[0160] For each titer assessment, a non template control (wells
containing all components except plasmid or genomic DNA) and a
negative control (all components including equivalent genomic DNA
from non-transduced PC-3 cells), is performed in triplicate. The
titer values are expressed in transduction units per milliliter
(TU/mL).
[0161] The potency of the vector of the disclosure is dependent on
both the replication of the vector and the resultant cytosine
deaminase (CD) activity in target cells. Therefore the potency
assay measures the increase in CD activity over time as vector
infection spreads in a previously uninfected cell line in tissue
culture. The assay measures the enzymatic activity of the
transferred yCD2 protein in transduced cells during early, middle
and late stages of infection by monitoring the conversion of
5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), using reverse
phase HPLC separation with UV detection. The increase of CD
activity over the course of the infection is a function of the
percent of cells infected over time and indicative of the TOCA 511
vector's ability to replicate. CD activity based on the 5-FC to
5-FU conversion rate is measured for each time point in CD units
per mg of protein (the specific activity, SA). The increase in SA
is then plotted over time, and reflects both the increase in the
percentage of cells transduced as a result of viral replication in
the culture, and the resultant transfer of CD activity. Accumulated
data from multiple assays and vector lots has been used to
determine an appropriate specification for this increase in SA of
CD, for product release. The assay has 1, 3 and 5 day timepoints
after an initial infection at an MOI of about 0.1 and a
non-infected control.
[0162] CD activity from late stage infected cells (day 5 time
point) was compared between lots to evaluate the use of this
activity as an Identity test. The assay includes the following
steps:
[0163] Transductions.
[0164] Transductions are performed in multi-well plate format on
U87 cells. For each transduction, three suitable dilutions are used
and each performed in triplicate. Cells are harvested at 0, 1, 3
and 5 days post transduction.
[0165] Set-Up of CD Reaction.
[0166] Cells are lysed and the total protein concentration
determined using the BCA protein assay using BSA as a standard. For
the yCD2 enzyme assay, an appropriate amount of cell lysate is
added to buffer containing 5-FC such that the rate of 5-FU
formation remains linear over 1-2 hours at 37.degree. C. The final
volume for the reaction mixture is 100 .mu.L. After 2 h, the enzyme
reaction is terminated by the addition of trichloroacetic acid,
briefly vortexed and prepared for subsequent HPLC analyses. Cell
lysates from non-transduced cells are used as a negative control
while a similar assay using samples from 100% infected cells is
used as a positive control.
[0167] HPLC Analysis.
[0168] The terminated reaction mixture is centrifuged at 12,000 rpm
for 5 minutes at room temperature in a micro-centrifuge. The
supernatant is then decanted away from the pellet and passed
through a 0.2p filter to further remove particulates before
injection onto a reverse phase HPLC column previously equilibrated
with an aqueous based mobile phase containing phosphate buffer at a
pH around 4.0. The chromatograms are followed at 260 nm and 280 nm
to monitor both substrate consumption and product formation.
Concentrations of either substrate or product are determined using
the graphing and analysis capabilities of GraphPad by comparing
them to previously generated standard curves calculated from known
substrate or product concentrations. Amounts of 5-FU generated over
1-2 h are used to determine CD units of activity (1 unit of CD
activity is defined as the formation 1 nmol of 5-FU per min) and
the Specific Activity is calculated dividing this number by the
amount of protein (from the cell lysate) used in the assay.
Example 7
Construction and Use of a Vector Encoding a Single Chain Antibody
to CTLA-4 (CD 152)
[0169] Single chain antibodies are derived from known full antibody
sequences that have a desired effect. Such sequences are available
(e.g. WO2006048749, US2006165706, U.S. Pat. No. 7,034,121, Genbank
Accession Numbers DJ437648, CS441506, CS441500, CS441494, CS441488,
the disclosures of which are incorporated herein by reference).
Such conventional antibody gene sequences are converted into single
chain antibody (scFv) sequences by commonly used methods known to
those skilled in the art (see for example Gilliland et al. "Rapid
and reliable cloning of antibody variable regions and generation of
recombinant single chain antibody fragments." Tissue Antigens 47,
1-20, 1996). Phage single chain antibodies to CTLA-4 are also
available from screening phage-scFv libraries directly (Pistillo et
al. Tissue Antigens 55:229 2000), and can be used directly for
insertion into the replicating retroviral vectors of the
disclosure. Regardless of how the sequence is derived, scFv are
typically about 700-900 bp in length and are synthesized by a
commercial vendor (BioBasic) with a PsiI site at the 5' end and
compatible NotI site at the 3' end, as described previously. This
sequence is then inserted into the replicating retroviral back bone
from pAC3-yCD2 at the PsiI-NotI sites after removal of the yCD2
sequence. Vector is produced and titered as described, and further
purified if necessary as described above. Human and Mouse CTLA4 are
very homologous in sequence and the replicating retrovirus of the
disclosure is first tested in a suitable syngeneic immunocompetent
mouse models such as the CT26/BALB/c model and S91 mouse melanoma
models, well known to those skilled in the art (see for example
Hodge et al J. Immunol. 174:5994 2005). Outcome is measured by one
or more of: modulation of tumor growth; lack of toxicity;
generation of antitumor responses; shrinkage of remote lesions
indicating systemic immunity. Doses are in the range of 10.sup.3 to
10.sup.7 TU in mice. In patients the vector is administered by
intralesional injection into tumor, or by administration into the
circulation that then carries the virus to the tumor. Doses are in
the range of 10.sup.5 to 10.sup.11 TU.
Example 8
Anti-Melanoma Efficacy Studies with Anti CD152 Single Chain
Antibody Expressing Vector in a Mouse Melanoma Model
[0170] Objective.
[0171] The objective of this study is to assess the effect of a
novel MLV based replication-competent retroviral vector carrying
single chain antibody directed against Cytotoxic T-Lymphocyte
Antigen 4 (CTLA-4) also referred to as Cluster of differentiation
152 (CD152) sequence (pAC3-.alpha.CD152) on melanoma growth, when
delivered via intratumoral (IT) injection in DBA/2 mice bearing
subcutaneous melanoma (Cloudman S91).
[0172] Mice.
[0173] Female DBA/2 or BALB/c mice (age .about.8 weeks) are
purchased from Jackson Laboratories (Bar Harbor, Me.). Mice are
acclimated for 7 days after arrival before start of studies.
[0174] Cells.
[0175] Cloudman S91 cells (ATCC, Manassas Va.) are a spontaneously
arising melanoma derived from DBA/2 mice. Cells are cultured in
Dulbecco's modified Eagles medium with 10% fetal bovine serum,
sodium pyruvate, and Glutamax (Hyclone, Logan Utah, and Invitrogen,
San Diego Calif.). Cells are resuspended in PBS (Hyclone, Logan
Utah) for implantation. S91 cells (1E6 in 100 .mu.L) are injected
into the right flank of DBA/2 mice.
[0176] Vector.
[0177] Vectors preparations are made by transient transfection (or
from a producer cell line in HT1080 cells) with titers of
approximately 7E6TU/ml. For initial studies vector is not further
purified or concentrated. For follow on experiments to determine
full dose response curves, high titer purified material is prepared
with a titer expected around 10.sup.8/ml. Vector is administered IT
in a volume of 50-100 .mu.L and IV in 100 .mu.L the total
dose/mouse of approximately 7E5 to 7E6 to 7ETU/mouse. Vector
expressing .alpha.CD152 is identified as Toca .alpha.CD152.
[0178] Tumor Implantation and Vector Injection.
[0179] Five groups of female DBA/2 (55 mice, 9-10 weeks of age) are
implanted subcutaneously with S91 melanoma cells (Day 0) and then
dosed (day 4-7 depending on growth rate of the S91 tumor;
approximately 50-100 mm.sup.3) with vehicle (Groups 1), with
control vector [AC3-GFP(V), (Group2), intratumor (IT) Toca
.alpha.CD152 vector injection (Groups 3), or intravenous Toca
.alpha.CD152 vector injection (group 4). Group 5 mice have no tumor
implanted and are intravenously injected with Toca .alpha.CD152
only.
[0180] Data Analysis.
[0181] Tumor growth analysis is carried out to 2000 mm.sup.3 or to
60 days based on which ever comes first. 10 mice from each group
will be plotted for tumor size over time. Statistical significance
will be determined using analysis of variance (ANOVA). P values of
<0.05 are considered statistically significant in all analyses,
which are performed with Prism 5 statistical software (GraphPad
Software) or equivalent. In-life observations are also taken to
assess any adverse events to .alpha.CD152 expression during
treatment.
[0182] Results.
[0183] Delivery of .alpha.CD152 by replicating MLV IT shows a
statistically significant retardation of growth compared to the
controls. Delivery of .alpha.CD152 by replicating MLV intravenously
shows a statistically significant retardation of growth compared to
the controls abrogates melanoma burden from the DBA/2--Cloudman S91
mouse melanoma model. Further animal studies were performed as
described more fully below.
Example 9
AC3-yCD2 Viral Vector is Therapeutic in an Intracranial Human
Xenograft (U87) in Nude Mice
[0184] An intracranial xenograft model using the U87 human glioma
cell line was established to test RCR vector spread and
biodistribution as well as therapeutic efficacy of RCR-vector
mediated cytosine deaminase suicide gene therapy in a nude mouse
host.
[0185] Following acclimation, mice were randomly assigned to one of
8 Treatment groups (see group description below). Seven groups
underwent intracranial administration into the right striatum of
1.times.10.sup.5 U87 cells administered/mouse on Day 0. Group 8
mice were not implanted with tumor. At Day 5, mice were injected
with Formulation Buffer only, or an RCR vector at
9.times.10.sup.5/5 .mu.l, 9.times.10.sup.4/5 .mu.l, or
9.times.10.sup.3/5 .mu.l. Mice receiving no vector, or vector at
9.times.10.sup.5/5 .mu.l or 9.times.10.sup.3/5 .mu.l were
randomized to receive 5-FC (500 mg/kg/day), administered as a
single IP injection, beginning on Day 19, or no 5-FC. Mice
receiving vector at mid dose all received 5-FC (i.e., No separate
control group for this dose). 5-FC administration continued daily
for 7 consecutive days followed by 15 days of no treatment. Cycles
of drug plus rest were repeated up to 4 cycles. 10 mice from each
group except group 8 were randomly assigned to the survival
analysis category. The remaining mice were sacrificed according to
a predetermined schedule.
TABLE-US-00018 Group Assignments and Dose Levels N per Analysis
Category Test Drug (A)Survival (B)Scheduled Group article Volume TX
N analysis Sacrifice 1 Form 5 .mu.l none 4 4 before buffer first
drug cycle 2 Form 5 .mu.l 5-FC 10 10 buffer 3 T5.0002 9e5/5 .mu.l
PBS 10 10 4 T5.0002 9e5/5 .mu.l 5FC 25 10 3 before start of each
cycle, 15 total 5 T5.0002 9e4/5 .mu.l 5FC 10 10 6 T5.0002 9e3/5
.mu.l 5FC 25 10 3 before start of each cycle, 15 total 7 T5.0002
9e3/5 .mu.l PBS 10 10 8 NO none 5FC 15 3 before TUMOR start of each
cycle, 15 total Total Number of Animals 109 60 49
[0186] Intravenous dosing was done via injection into the tail
vein. Intraperitoneal dosing was done via injection into the
abdomen with care taken to avoid the bladder. For intracranial
injection mice were anesthetized with isoflurane and positioned in
a stereotaxic device with blunt ear bars. The skin was shaved and
betadine was used to treat the scalp to prepare the surgical site.
The animal was placed on a heating pad and a scalpel used under
sterile conditions to make a midline incision through the skin.
Retraction of the skin and reflection of the fascia at the incision
site will allow for visualization of the skull. A guide cannula
with a 3 mm projection, fitted with a cap with a 3.5 mm projection,
will be inserted through a small burr hole in the skull and
attached with dental cement and three small screws to the skull.
After hardening of the cement, the skin will be closed with
sutures. The projected stereotaxic coordinates are AP=0.5-1.0 mm,
ML=1.8-2.0 mm, DV=3.0 mm. Exact stereotaxic coordinates for the
cohort of animals received will be determined in a pilot experiment
(2-3 animals) by injecting dye and determining its location. The
animals will be monitored during anesthesia recovery. Analgesics,
buprenorphine, will be administered subcutaneously (SC) before the
end of the procedure then buprenorphine will be administered
approximately every 12 hrs for up to 3 days. Animals will be
monitored on a daily basis. Cells or vector were intracranially
infused through an injection cannula with a 3.5 mm projection
inserted through the guide cannula. The rate was controlled with a
syringe pump fitted with a Hamilton syringe and flexible tubing.
For cell injection, 1 microliter of cells was delivered at a flow
rate of 0.2 microliters per minute (5 minutes total). For vector
injection, 5 microliters of vector was delivered at a flow rate 0f
0.33 microliters per minute (15 minutes total).
[0187] Vector was delivered and calculated as transforming units
(TU) per gram of brain weight to the mice. Using such calculation
the translation of dose can be calculated for other mammals
including humans. FIG. 8 shows the effect on vector dose in this
mouse model.
Example 10
AC3-yCD2(V) is Therapeutic in a Syngeneic Mouse Model of Brain
Cancer
[0188] An intracranial implant model using the CT26 colorectal
cancer cell line in syngeneic BALB/c mice was established to test
RCR vector spread and biodistribution as well as therapeutic
efficacy of RCR-vector mediated cytosine deaminase suicide gene
therapy and its immunological impact.
[0189] This study included 129 animals, 0 Male, 119 Female and 10
contingency animals (10 Female). Following acclimation, mice were
randomly assigned to one of 8 Treatment groups (see group
description below). Seven groups underwent intracranial
administration into the right striatum of 1.times.10.sup.4 CT26
cells administered/mouse on Day 0. Group 8 mice were not implanted
with tumor. At Day 4, mice were injected with Formulation Buffer
only, or vector at 9.times.10.sup.5/5 .mu.l, 9.times.10.sup.4/5
.mu.l, or 9.times.10.sup.3/5 .mu.l. Mice receiving no vector, or
vector at 9.times.10.sup.5/5 .mu.l or 9.times.10.sup.3/5 .mu.l were
randomized to receive 5-FC (500 mg/kg/BID), administered by IP
injection, beginning on Day 13, or no 5-FC. Mice receiving vector
at mid dose received 5-FC (ie. No separate control group for this
dose). 5-FC administration continued daily for 7 consecutive days
followed by 10 days of no treatment. Cycles of drug plus rest were
repeated up to 4 cycles. 10 mice from each group except group 8
were randomly assigned to the survival analysis category. The
remaining mice were sacrificed according to a predetermined
schedule.
[0190] Naive sentinel mice were co-housed with the scheduled
sacrifice animals and taken down at the same time points to assess
vector transmittal through shedding.
TABLE-US-00019 Group Assignments and Dose Levels N per Analysis
Category Test Drug (A)Survival (B)Scheduled (C) Group article
Volume TX N analysis Sacrifice Sentinels 1 Form 5 .mu.l PBS 4 4
before buffer first drug cycle 2 Form 5 .mu.l 5FC 10 10 buffer 3
T5.0002 9E5/5 .mu.l PBS 10 10 4 T5.0002 9E5/5 .mu.l 5FC 25 10 3
before 1 before start of each start of each cycle, 15 cycle, 5
total total 5 T5.0002 9E4/5 .mu.l 5FC 10 10 6 T5.0002 9E3/5 .mu.l
5FC 25 10 3 before 1 before start of each start of each cycle, 15
cycle, 5 total total 7 T5.0002 9E3/5 .mu.l PBS 10 10 8 NO none 5FC
15 3 before TUMOR start of each cycle, 15 total Total Number of
Animals 119 60 49 10
[0191] Intravenous dosing was done via injection into the tail
vein. Intraperitoneal dosing was done via injection into the
abdomen with care taken to avoid the bladder. For intracranial
administration, mice with a guide cannula with a 3.2 mm projection
implanted into the right striatum, and fitted with a cap with a 3.7
mm projection were used. The projected stereotaxic coordinates are
AP=0.5-1.0 mm, ML=1.8-2.0 mm, DV=3.2 mm (from bregma). Cells or
vector were intracranially infused through an injection cannula
with a 3.7 mm projection inserted through the guide cannula. The
rate was controlled with a syringe pump fitted with a Hamilton
syringe and flexible tubing.
[0192] For cell injection, 1 microliter of cells was delivered at a
flow rate of 0.2 microliter per minute (5 minutes total). For
vector injection, 5 microliter of vector was delivered at a flow
rate of 0.33 microliter per minute (15 minutes total).
[0193] Vector was delivered and calculated as transforming units
(TU) per gram of brain weight to the mice. Using such calculation
the translation of dose can be calculated for other mammals
including humans. FIG. 9 shows the effect on vector dose in this
mouse model when the vector is delivered intracranially.
Example 11
Construction and Testing of RCR Vectors Expressing miR-128-1 and
miR128-2
[0194] Construction of Recombinant Replication Competent Retroviral
Vector Containing a Heterologous Polynucleotide Sequence of Human
pri-miRNA-128-1.
[0195] The replication competent retroviral vector, pAC3-miR-128-1
expressing miR-128-1 was derived from the backbone of pAC3-yCD2
described in one of the embodiments. The pAC3 backbone in the
pAC3-miR-128-1 vector was isolated by endonuclease digestion of the
pAC3-yCD2 plasmid DNA with Mlu I and Not I to remove the IRES-yCD2
polynucleotide sequence. The polynucleotide DNA sequence of
pri-miR-128-1 was obtained from the product sheet of the
pEP-mir-128-1 expression vector (Cell BioLabs Inc.) (SEQ ID NO:
31). DNA sequence of pri-miR-128-1 was synthesized with a Mlu I
restriction enzyme site at the 5' end and a Not I restriction
enzyme site at the 3' end of the double-stranded DNA fragment for
subsequent insertion at the corresponding site in the Mlu I and Not
I digested pAC3-yCD2 plasmid DNA described above. The resulting
construct, pAC3-miR-128-1, encodes 3 genes: the gag, the pol, and
the env, and the non-coding pri-miR-128-1 sequence.
[0196] Testing of Expression of Mature miR-128 from Cells
Transduced with miR-128 Containing Recombinant Replication
Competent Retroviral Vector.
[0197] In order to confirm the expression of miR-128 from cells
transduced with miR-128 containing recombinant replication
competent retroviral vectors, cells from day 9 post infection at
which the maximal infectivity has reached were expanded and
harvested to extract total RNA for detection of mature miRNA
expression. The results from Taqman microRNA assay showed an over
expression of mature miR-128 from both HT1080 and U87-MG cells
transduced with pAC3-miR-128-1, pAC3-miR-128-2, and
pAC3-H1-shRNAmiR128 vectors, respectively, compared to untransduced
cells. In both cell lines, cells transduced with pAC3-miR-128-1 and
pAC3-H1-shRNAmiR128 vector expressed higher level of mature miR-128
than cells transduced with pAC3-miR-128-2 vector.
Example 12
Construction and Testing of Recombinant Replication Competent
Retroviral Vector Containing Heterologous Polynucleotide Sequences
of IRES, yCD2, Human H1 Promoter and Human Pre-miR128-1
[0198] Construction.
[0199] The replication competent retroviral vector,
pAC3-yCD2-H1-shRNAmiR128 is derived from the backbone of pAC3-yCD2
described in one of the embodiments. The pAC3-yCD2 backbone in the
pAC3-yCD2-H1-shRNAmiR128 vector is isolated by endonuclease
digestion of the pAC3-yCD2 plasmid DNA with Not I. The
polynucleotide DNA sequence of the human H1 promoter is obtained
from the product information of pSilencer 3.1 H1 hygro expression
vector (Ambion), and the polynucleotide DNA sequence of the short
hairpin structured pre-miR-128-1 is obtained from the
http:(//)www.mirbase.org/. DNA sequence of pre-miR128-1 linked to
the human H1 promoter (SEQ ID NO: 34) is synthesized with a Not I
restriction enzyme site at both ends of the double-stranded DNA
fragment for subsequent insertion at the corresponding site in Not
I digested pAC3-yCD2 plasmid DNA described above. The resulting
construct, pAC3-H1-shRNAmiR128, encodes 4 genes: the gag, the pol,
and the env, and the yCD2, and the non-coding short hairpin
structured pre-miR-128-1 sequence.
[0200] Vector stock is produced by transient transfection of the
vector-encoding plasmid DNA into 293T cells using calcium phosphate
method. Eighteen hours post transfection, the culture is replaced
with fresh medium. Twenty-four hours post medium replacement, the
supernatant containing the vector is collected and filtered through
a 0.45 .mu.m filter and used immediately or stored in aliquots at
-80.degree. C. for later use. Twenty micro-liter of the collected
vector stocks is used to infect human prostate cancer cells, PC3.
Twenty-four hours post infection, AZT is added to the cells to
inhibit further viral replication. Forty-eight hours post
infection, genomic DNA of infected PC3 cells is extracted for titer
assay. The titer of the vector stocks is determined by qPCR with an
inclusion of standards of known copy numbers.
[0201] Testing of Replication Kinetics of the
pAC3-yCD2-H1-shRNAmiR128 Recombinant Replication Competent
Retroviral Vectors in Culture.
[0202] In order to confirm that the incorporation of
H1-pre-miR-128-1 replicates normally, calculated volume of each
vector stocks collected from transient transfection mentioned above
is used to infect fresh human fibrosarcoma cells, HT1080 and human
glioma cells, U87-MG, respectively, at a MOI of 0.1. Transduced
cells are passaged at day 3, 6 and 9 post infection. At each time
point, a portion of cells are collected for genomic DNA extraction
for qPCR. Dilutions of genomic DNA are made to generate aliquots of
genomic DNA with same concentration for equal amount of genomic
in-put in qPCR. Replication kinetics of each vectors are generated
by plotting inversed C(t) values vs. time points. Result show that
the vector replicates at similar kinetics compared to control MLV
virus.
[0203] Testing of Expression of Mature miR-128 from Cells
Transduced with the pAC3-yCD2-H1-shRNAmiR128 Recombinant
Replication Competent Retroviral Vector.
[0204] To confirm the expression of miR-128 from cells transduced
with pAC3-yCD2-H1-shRNAmiR128 recombinant replication competent
retroviral vector, cells from day 9 post infection, at which the
maximal infectivity is reached, are expanded and harvested to
extract total RNA for detection of mature miRNA expression. Result
from Taqman microRNA assay shows an over expression of mature
miR-128 from both HT1080 and U87-MG cells transduced with the
pAC3-yCD2-H1-shRNAmiR128compared to untransduced cells.
[0205] Testing of Bmi-1 Expression from Cells Transduced with
pAC3-yCD2-H1-shRNAmiR128 Recombinant Replication Competent
Retroviral Vectors to Demonstrate Target Engagement of miR-128.
[0206] Bmi-1 expression has been observed to be up-regulated in a
variety of human cancers including glioblastoma, and has been shown
to be the target of miR-128. To confirm target engagement of
miR-128, Bmi-1 expression from cells transduced with
pAC3-yCD2-H1-shRNAmiR128 is detected by qRT-PCR. The result shows
that U87-MG cells transduced with pAC3-yCD2-H1-shRNAmiR128 express
lower level of Bmi-1 than untransduced cells, whereas in HT1080
cells no significant difference was observed between transduced and
untransduced cells. The data support the concept that miR-128 plays
an important functional role in the central nervous system.
[0207] Testing of yCD2 Expression from Cells Transduced with
pAC3-yCD2-H1-shRNAmiR128 by Immune-Blot.
[0208] To confirm the expression of yCD2 from cells transduced with
pAC3-yCD2-H1-shRNAmiR128 recombinant replication competent
retroviral vector, cells from day 9 post infection, at which the
maximal infectivity is reached, are expanded and harvested to
extract total protein for detection of yCD2 expression. The result
from immune-blot shows normal expression yCD2 from both HT1080 and
U87-MG cells transduced with the pAC3-yCD2-H1-shRNAmiR128 compared
to pAC3-yCD2 transduced cells.
Example 13
Anti-Tumor Efficacy Studies with miRNA Expressing Vector in a
Mouse/Human Xenograft Model
[0209] Objective.
[0210] The objective of this study is to assess the effect of a
novel MLV based replication-competent retroviral vectors carrying
the miR128 sequence (AC3-miR128-1(V); AC3-miR128-2(V);
AC3-miR128-3(V) on survival, when delivered via intracranial (IC)
injection in nude mice bearing a human glioma xenograft, at three
Toca 511 dose levels.
[0211] Mice.
[0212] Female athymic nude-Foxn1 nu (nude) mice (age .about.8
weeks) are purchased from Harlan (Indianapolis Ind.). Mice are
acclimated for 7 days after arrival. Mice undergo surgical
placement of an indwelling guide cannula with a 3.0 mm projection
implanted into the right striatum, and fitted with a cap containing
a 3.5 mm projection. The stereotaxic coordinates are AP=+0.5 mm,
ML=-1.8 mm (from bregma).
[0213] Cells.
[0214] U-87 MG cells (ATCC, Manassas Va.) are derived from a
malignant glioma from a 44 year old Caucasian female. Cells are
cultured in Dulbecco's modified Eagles medium with 10% fetal bovine
serum, sodium pyruvate, and Glutamax (Hyclone, Logan Utah, and
Invitrogen, San Diego Calif.). Cells are resuspended in PBS
(Hyclone, Logan Utah) for implantation. U-87 MG cells (1E5 in 1
.mu.L) are infused at 0.2 .mu.L per minute (5 minutes, followed by
a hold of 5 minutes) IC through an injection cannula with a 3.5 mm
projection inserted through the guide cannula.
[0215] Vectors preparations are made by transient transfection (or
from a producer cell line) and all have titers of approximately
5E6TU/ml. For initial studies vector is not further purified or
concentrated. For follow on experiments to determine full dose
response curves, high titer purified material is prepared with a
titer of around 10E8/ml. Vector is administered IC in a volume of 5
ul or less for a minimum total dose/mouse of approximately 2.5E4
TU/mouse.
[0216] Tumor Implantation and Vector Injection.
[0217] Six groups of female athymic nude-Foxn1 nu mice (65 mice,
9-10 weeks of age) are implanted IC with U-87 tumor cells (Day 0)
then dosed IC (day 4-7 depending on growth rate of the U87 cells)
with vehicle (Groups 1), with control vector (AC3-GFP(V), Group2)
or IC with AC3-miR128-1(V); AC3-miR128-2(V); AC3-miR128-3(V)
(Groups 3-5). Group 6 mice were not implanted with tumor or
vector.
[0218] Data Analysis.
[0219] Survival analysis to day 60 is performed on 10 mice each
from Groups 1-6 and plotted as a Kaplan Meyer plot. Survival curves
are compared by the log-rank test. P values of <0.05 are
considered statistically significant in all analyses, which are
performed with Prism 5 statistical software (GraphPad Software) or
equivalent.
[0220] Results from treatment with the vectors show a statistically
significant survival advantage in this human glioma xenograft model
compared to treatment with control vector or vehicle alone.
Example 14
Thymosin Combination Therapy
[0221] Experiments using Thymosin Alpha 1 were performed in
conjunction with Toca 511 treatment in the Tu2449/B6C3F1 glioma
mouse model (U. Pohle et al. Int J Oncol. 15:829-834 (1999); HM.
Smilowitz et al. J. Neurosurg 106:652-659 2007). Experiments were
conducted in a similar manner to those in the BALB/c-CT26 model
(Example 10), except that the initial intracranial cell innoculum
was at 10.sup.4 cells and the 5-FC dosing was twice a day (BID)
intra-peritoneally at 500 mg/kg, with 10 days off drug followed by
4 days with 5-FC administration. In addition to the administration
of vector and 5-FC some groups were dosed with thymosin alpha
1(TA1). Thymosin Alpha 1 was obtained from Sigma Aldrich cat# T3641
and a stock solution made in sterile water at 400 .mu.g/mL. TA1
(200 .mu.g/kg, .about.40 .mu.g/animal) was given IP starting on day
7 SID for 28 days.
[0222] The results are presented in FIG. 10. Using a suboptimal
dose of Toca511 (E3) and standard BID 5-FC dosing, the addition of
thymosin alpha 1 increased the survival rate to that of the optimal
E5 dosing of Toca 511. When compared to buffer only animals there
was a significant survival advantage (p=0.0016, hazard ratio
0.1111, 95% CL 0.028 to 0.436). Further, using a suboptimal dose of
Toca511 (E3), thymosin alpha 1 dosing, and standard BID 5-FC dosing
resulted in a survival advantage compared to a suboptimal dose of
Toca511 (E3) and only thymosin alpha 1 dosing.
[0223] A number of embodiments of the disclosure have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the disclosure. Accordingly, other embodiments are within
the scope of the following claims.
Sequence CWU 1
1
731477DNASaccharomyces cerevisiaeCDS(1)..(477) 1atg gtg aca ggg gga
atg gca agc aag tgg gat cag aag ggt atg gac 48Met Val Thr Gly Gly
Met Ala Ser Lys Trp Asp Gln Lys Gly Met Asp 1 5 10 15 att gcc tat
gag gag gcg gcc tta ggt tac aaa gag ggt ggt gtt cct 96Ile Ala Tyr
Glu Glu Ala Ala Leu Gly Tyr Lys Glu Gly Gly Val Pro 20 25 30 att
ggc gga tgt ctt atc aat aac aaa gac gga agt gtt ctc ggt cgt 144Ile
Gly Gly Cys Leu Ile Asn Asn Lys Asp Gly Ser Val Leu Gly Arg 35 40
45 ggt cac aac atg aga ttt caa aag gga tcc gcc aca cta cat ggt gag
192Gly His Asn Met Arg Phe Gln Lys Gly Ser Ala Thr Leu His Gly Glu
50 55 60 atc tcc act ttg gaa aac tgt ggg aga tta gag ggc aaa gtg
tac aaa 240Ile Ser Thr Leu Glu Asn Cys Gly Arg Leu Glu Gly Lys Val
Tyr Lys 65 70 75 80 gat acc act ttg tat acg acg ctg tct cca tgc gac
atg tgt aca ggt 288Asp Thr Thr Leu Tyr Thr Thr Leu Ser Pro Cys Asp
Met Cys Thr Gly 85 90 95 gcc atc atc atg tat ggt att cca cgc tgt
gtt gtc ggt gag aac gtt 336Ala Ile Ile Met Tyr Gly Ile Pro Arg Cys
Val Val Gly Glu Asn Val 100 105 110 aat ttc aaa agt aag ggc gag aaa
tat tta caa act aga ggt cac gag 384Asn Phe Lys Ser Lys Gly Glu Lys
Tyr Leu Gln Thr Arg Gly His Glu 115 120 125 gtt gtt gtt gtt gac gat
gag agg tgt aaa aag atc atg aaa caa ttt 432Val Val Val Val Asp Asp
Glu Arg Cys Lys Lys Ile Met Lys Gln Phe 130 135 140 atc gat gaa aga
cct cag gat tgg ttt gaa gat att ggt gag tag 477Ile Asp Glu Arg Pro
Gln Asp Trp Phe Glu Asp Ile Gly Glu 145 150 155
2158PRTSaccharomyces cerevisiae 2Met Val Thr Gly Gly Met Ala Ser
Lys Trp Asp Gln Lys Gly Met Asp 1 5 10 15 Ile Ala Tyr Glu Glu Ala
Ala Leu Gly Tyr Lys Glu Gly Gly Val Pro 20 25 30 Ile Gly Gly Cys
Leu Ile Asn Asn Lys Asp Gly Ser Val Leu Gly Arg 35 40 45 Gly His
Asn Met Arg Phe Gln Lys Gly Ser Ala Thr Leu His Gly Glu 50 55 60
Ile Ser Thr Leu Glu Asn Cys Gly Arg Leu Glu Gly Lys Val Tyr Lys 65
70 75 80 Asp Thr Thr Leu Tyr Thr Thr Leu Ser Pro Cys Asp Met Cys
Thr Gly 85 90 95 Ala Ile Ile Met Tyr Gly Ile Pro Arg Cys Val Val
Gly Glu Asn Val 100 105 110 Asn Phe Lys Ser Lys Gly Glu Lys Tyr Leu
Gln Thr Arg Gly His Glu 115 120 125 Val Val Val Val Asp Asp Glu Arg
Cys Lys Lys Ile Met Lys Gln Phe 130 135 140 Ile Asp Glu Arg Pro Gln
Asp Trp Phe Glu Asp Ile Gly Glu 145 150 155 3477DNAArtificial
SequenceEngineered cytosine deaminase 3atg gtg aca ggg gga atg gca
agc aag tgg gat cag aag ggt atg gac 48Met Val Thr Gly Gly Met Ala
Ser Lys Trp Asp Gln Lys Gly Met Asp 1 5 10 15 att gcc tat gag gag
gcg tta tta ggt tac aaa gag ggt ggt gtt cct 96Ile Ala Tyr Glu Glu
Ala Leu Leu Gly Tyr Lys Glu Gly Gly Val Pro 20 25 30 att ggc gga
tgt ctt atc aat aac aaa gac gga agt gtt ctc ggt cgt 144Ile Gly Gly
Cys Leu Ile Asn Asn Lys Asp Gly Ser Val Leu Gly Arg 35 40 45 ggt
cac aac atg aga ttt caa aag gga tcc gcc aca cta cat ggt gag 192Gly
His Asn Met Arg Phe Gln Lys Gly Ser Ala Thr Leu His Gly Glu 50 55
60 atc tcc act ttg gaa aac tgt ggg aga tta gag ggc aaa gtg tac aaa
240Ile Ser Thr Leu Glu Asn Cys Gly Arg Leu Glu Gly Lys Val Tyr Lys
65 70 75 80 gat acc act ttg tat acg acg ctg tct cca tgc gac atg tgt
aca ggt 288Asp Thr Thr Leu Tyr Thr Thr Leu Ser Pro Cys Asp Met Cys
Thr Gly 85 90 95 gcc atc atc atg tat ggt att cca cgc tgt gtc atc
ggt gag aac gtt 336Ala Ile Ile Met Tyr Gly Ile Pro Arg Cys Val Ile
Gly Glu Asn Val 100 105 110 aat ttc aaa agt aag ggc gag aaa tat tta
caa act aga ggt cac gag 384Asn Phe Lys Ser Lys Gly Glu Lys Tyr Leu
Gln Thr Arg Gly His Glu 115 120 125 gtt gtt gtt gtt gac gat gag agg
tgt aaa aag tta atg aaa caa ttt 432Val Val Val Val Asp Asp Glu Arg
Cys Lys Lys Leu Met Lys Gln Phe 130 135 140 atc gat gaa aga cct cag
gat tgg ttt gaa gat att ggt gag tag 477Ile Asp Glu Arg Pro Gln Asp
Trp Phe Glu Asp Ile Gly Glu 145 150 155 4158PRTArtificial
SequenceSynthetic Construct 4Met Val Thr Gly Gly Met Ala Ser Lys
Trp Asp Gln Lys Gly Met Asp 1 5 10 15 Ile Ala Tyr Glu Glu Ala Leu
Leu Gly Tyr Lys Glu Gly Gly Val Pro 20 25 30 Ile Gly Gly Cys Leu
Ile Asn Asn Lys Asp Gly Ser Val Leu Gly Arg 35 40 45 Gly His Asn
Met Arg Phe Gln Lys Gly Ser Ala Thr Leu His Gly Glu 50 55 60 Ile
Ser Thr Leu Glu Asn Cys Gly Arg Leu Glu Gly Lys Val Tyr Lys 65 70
75 80 Asp Thr Thr Leu Tyr Thr Thr Leu Ser Pro Cys Asp Met Cys Thr
Gly 85 90 95 Ala Ile Ile Met Tyr Gly Ile Pro Arg Cys Val Ile Gly
Glu Asn Val 100 105 110 Asn Phe Lys Ser Lys Gly Glu Lys Tyr Leu Gln
Thr Arg Gly His Glu 115 120 125 Val Val Val Val Asp Asp Glu Arg Cys
Lys Lys Leu Met Lys Gln Phe 130 135 140 Ile Asp Glu Arg Pro Gln Asp
Trp Phe Glu Asp Ile Gly Glu 145 150 155 5480DNAArtificial
SequenceHuman codon optimized cytosine deaminase 5atg gtg acc ggc
ggc atg gcc tcc aag tgg gat caa aag ggc atg gat 48Met Val Thr Gly
Gly Met Ala Ser Lys Trp Asp Gln Lys Gly Met Asp 1 5 10 15 atc gct
tac gag gag gcc gca ctg ggc tac aag gag ggc ggc gtg cct 96Ile Ala
Tyr Glu Glu Ala Ala Leu Gly Tyr Lys Glu Gly Gly Val Pro 20 25 30
atc ggc ggc tgt ctg atc aac aac aag gac ggc agt gtg ctg ggc agg
144Ile Gly Gly Cys Leu Ile Asn Asn Lys Asp Gly Ser Val Leu Gly Arg
35 40 45 ggc cac aac atg agg ttc cag aag ggc tcc gcc acc ctg cac
ggc gag 192Gly His Asn Met Arg Phe Gln Lys Gly Ser Ala Thr Leu His
Gly Glu 50 55 60 atc tcc acc ctg gag aac tgt ggc agg ctg gag ggc
aag gtg tac aag 240Ile Ser Thr Leu Glu Asn Cys Gly Arg Leu Glu Gly
Lys Val Tyr Lys 65 70 75 80 gac acc acc ctg tac acc acc ctg tcc cct
tgt gac atg tgt acc ggc 288Asp Thr Thr Leu Tyr Thr Thr Leu Ser Pro
Cys Asp Met Cys Thr Gly 85 90 95 gct atc atc atg tac ggc atc cct
agg tgt gtg gtc ggc gag aac gtg 336Ala Ile Ile Met Tyr Gly Ile Pro
Arg Cys Val Val Gly Glu Asn Val 100 105 110 aac ttc aag tcc aag ggc
gag aag tac ctg caa acc agg ggc cac gag 384Asn Phe Lys Ser Lys Gly
Glu Lys Tyr Leu Gln Thr Arg Gly His Glu 115 120 125 gtg gtg gtt gtt
gac gat gag agg tgt aag aag atc atg aag cag ttc 432Val Val Val Val
Asp Asp Glu Arg Cys Lys Lys Ile Met Lys Gln Phe 130 135 140 atc gac
gag agg cct cag gac tgg ttc gag gat atc ggc gag tga taa 480Ile Asp
Glu Arg Pro Gln Asp Trp Phe Glu Asp Ile Gly Glu 145 150 155
6158PRTArtificial SequenceSynthetic Construct 6Met Val Thr Gly Gly
Met Ala Ser Lys Trp Asp Gln Lys Gly Met Asp 1 5 10 15 Ile Ala Tyr
Glu Glu Ala Ala Leu Gly Tyr Lys Glu Gly Gly Val Pro 20 25 30 Ile
Gly Gly Cys Leu Ile Asn Asn Lys Asp Gly Ser Val Leu Gly Arg 35 40
45 Gly His Asn Met Arg Phe Gln Lys Gly Ser Ala Thr Leu His Gly Glu
50 55 60 Ile Ser Thr Leu Glu Asn Cys Gly Arg Leu Glu Gly Lys Val
Tyr Lys 65 70 75 80 Asp Thr Thr Leu Tyr Thr Thr Leu Ser Pro Cys Asp
Met Cys Thr Gly 85 90 95 Ala Ile Ile Met Tyr Gly Ile Pro Arg Cys
Val Val Gly Glu Asn Val 100 105 110 Asn Phe Lys Ser Lys Gly Glu Lys
Tyr Leu Gln Thr Arg Gly His Glu 115 120 125 Val Val Val Val Asp Asp
Glu Arg Cys Lys Lys Ile Met Lys Gln Phe 130 135 140 Ile Asp Glu Arg
Pro Gln Asp Trp Phe Glu Asp Ile Gly Glu 145 150 155
7756DNASaccharomyces cerevisiaeCDS(1)..(756) 7atg aac ccg tta ttc
ttt ttg gct tct cca ttc ttg tac ctt aca tat 48Met Asn Pro Leu Phe
Phe Leu Ala Ser Pro Phe Leu Tyr Leu Thr Tyr 1 5 10 15 ctt ata tat
tat cca aac aaa ggg tct ttc gtt agc aaa cct aga aat 96Leu Ile Tyr
Tyr Pro Asn Lys Gly Ser Phe Val Ser Lys Pro Arg Asn 20 25 30 ctg
caa aaa atg tct tcg gaa cca ttt aag aac gtc tac ttg cta cct 144Leu
Gln Lys Met Ser Ser Glu Pro Phe Lys Asn Val Tyr Leu Leu Pro 35 40
45 caa aca aac caa ttg ctg ggt ttg tac acc atc atc aga aat aag aat
192Gln Thr Asn Gln Leu Leu Gly Leu Tyr Thr Ile Ile Arg Asn Lys Asn
50 55 60 aca act aga cct gat ttc att ttc tac tcc gat aga atc atc
aga ttg 240Thr Thr Arg Pro Asp Phe Ile Phe Tyr Ser Asp Arg Ile Ile
Arg Leu 65 70 75 80 ttg gtt gaa gaa ggt ttg aac cat cta cct gtg caa
aag caa att gtg 288Leu Val Glu Glu Gly Leu Asn His Leu Pro Val Gln
Lys Gln Ile Val 85 90 95 gaa act gac acc aac gaa aac ttc gaa ggt
gtc tca ttc atg ggt aaa 336Glu Thr Asp Thr Asn Glu Asn Phe Glu Gly
Val Ser Phe Met Gly Lys 100 105 110 atc tgt ggt gtt tcc att gtc aga
gct ggt gaa tcg atg gag caa gga 384Ile Cys Gly Val Ser Ile Val Arg
Ala Gly Glu Ser Met Glu Gln Gly 115 120 125 tta aga gac tgt tgt agg
tct gtg cgt atc ggt aaa att tta att caa 432Leu Arg Asp Cys Cys Arg
Ser Val Arg Ile Gly Lys Ile Leu Ile Gln 130 135 140 agg gac gag gag
act gct tta cca aag tta ttc tac gaa aaa tta cca 480Arg Asp Glu Glu
Thr Ala Leu Pro Lys Leu Phe Tyr Glu Lys Leu Pro 145 150 155 160 gag
gat ata tct gaa agg tat gtc ttc cta tta gac cca atg ctg gcc 528Glu
Asp Ile Ser Glu Arg Tyr Val Phe Leu Leu Asp Pro Met Leu Ala 165 170
175 acc ggt ggt agt gct atc atg gct aca gaa gtc ttg att aag aga ggt
576Thr Gly Gly Ser Ala Ile Met Ala Thr Glu Val Leu Ile Lys Arg Gly
180 185 190 gtt aag cca gag aga att tac ttc tta aac cta atc tgt agt
aag gaa 624Val Lys Pro Glu Arg Ile Tyr Phe Leu Asn Leu Ile Cys Ser
Lys Glu 195 200 205 ggg att gaa aaa tac cat gcc gcc ttc cca gag gtc
aga att gtt act 672Gly Ile Glu Lys Tyr His Ala Ala Phe Pro Glu Val
Arg Ile Val Thr 210 215 220 ggt gcc ctc gac aga ggt cta gat gaa aac
aag tat cta gtt cca ggg 720Gly Ala Leu Asp Arg Gly Leu Asp Glu Asn
Lys Tyr Leu Val Pro Gly 225 230 235 240 ttg ggt gac ttt ggt gac aga
tac tac tgt gtt taa 756Leu Gly Asp Phe Gly Asp Arg Tyr Tyr Cys Val
245 250 8251PRTSaccharomyces cerevisiae 8Met Asn Pro Leu Phe Phe
Leu Ala Ser Pro Phe Leu Tyr Leu Thr Tyr 1 5 10 15 Leu Ile Tyr Tyr
Pro Asn Lys Gly Ser Phe Val Ser Lys Pro Arg Asn 20 25 30 Leu Gln
Lys Met Ser Ser Glu Pro Phe Lys Asn Val Tyr Leu Leu Pro 35 40 45
Gln Thr Asn Gln Leu Leu Gly Leu Tyr Thr Ile Ile Arg Asn Lys Asn 50
55 60 Thr Thr Arg Pro Asp Phe Ile Phe Tyr Ser Asp Arg Ile Ile Arg
Leu 65 70 75 80 Leu Val Glu Glu Gly Leu Asn His Leu Pro Val Gln Lys
Gln Ile Val 85 90 95 Glu Thr Asp Thr Asn Glu Asn Phe Glu Gly Val
Ser Phe Met Gly Lys 100 105 110 Ile Cys Gly Val Ser Ile Val Arg Ala
Gly Glu Ser Met Glu Gln Gly 115 120 125 Leu Arg Asp Cys Cys Arg Ser
Val Arg Ile Gly Lys Ile Leu Ile Gln 130 135 140 Arg Asp Glu Glu Thr
Ala Leu Pro Lys Leu Phe Tyr Glu Lys Leu Pro 145 150 155 160 Glu Asp
Ile Ser Glu Arg Tyr Val Phe Leu Leu Asp Pro Met Leu Ala 165 170 175
Thr Gly Gly Ser Ala Ile Met Ala Thr Glu Val Leu Ile Lys Arg Gly 180
185 190 Val Lys Pro Glu Arg Ile Tyr Phe Leu Asn Leu Ile Cys Ser Lys
Glu 195 200 205 Gly Ile Glu Lys Tyr His Ala Ala Phe Pro Glu Val Arg
Ile Val Thr 210 215 220 Gly Ala Leu Asp Arg Gly Leu Asp Glu Asn Lys
Tyr Leu Val Pro Gly 225 230 235 240 Leu Gly Asp Phe Gly Asp Arg Tyr
Tyr Cys Val 245 250 91443DNAhomo sapiensCDS(1)..(1443) 9atg gct gtt
gct cgt gct gct ctt ggt cct ctt gtt act ggt ctt tat 48Met Ala Val
Ala Arg Ala Ala Leu Gly Pro Leu Val Thr Gly Leu Tyr 1 5 10 15 gat
gtt caa gct ttt aaa ttt ggt gat ttt gtt ctt aaa tct ggt ctt 96Asp
Val Gln Ala Phe Lys Phe Gly Asp Phe Val Leu Lys Ser Gly Leu 20 25
30 tct tct cct att tat att gat ctt cgt ggt att gtt tct cgt cct cgt
144Ser Ser Pro Ile Tyr Ile Asp Leu Arg Gly Ile Val Ser Arg Pro Arg
35 40 45 ctt ctt tct caa gtt gct gat att ctt ttt caa act gct caa
aat gct 192Leu Leu Ser Gln Val Ala Asp Ile Leu Phe Gln Thr Ala Gln
Asn Ala 50 55 60 ggt att tct ttt gat act gtt tgt ggt gtt cct tat
act gct ctt cct 240Gly Ile Ser Phe Asp Thr Val Cys Gly Val Pro Tyr
Thr Ala Leu Pro 65 70 75 80 ctt gct act gtt att tgt tct act aat caa
att cct atg ctt att cgt 288Leu Ala Thr Val Ile Cys Ser Thr Asn Gln
Ile Pro Met Leu Ile Arg 85 90 95 cgt aaa gaa act aaa gat tat ggt
act aaa cgt ctt gtt gaa ggt act 336Arg Lys Glu Thr Lys Asp Tyr Gly
Thr Lys Arg Leu Val Glu Gly Thr 100 105 110 att aat cct ggt gaa act
tgt ctt att att gaa gat gtt gtt act tct 384Ile Asn Pro Gly Glu Thr
Cys Leu Ile Ile Glu Asp Val Val Thr Ser 115 120 125 ggt tct tct gtt
ctt gaa act gtt gaa gtt ctt caa aaa gaa ggt ctt 432Gly Ser Ser Val
Leu Glu Thr Val Glu Val Leu Gln Lys Glu Gly Leu 130 135 140 aaa gtt
act gat gct att gtt ctt ctt gat cgt gaa caa ggt ggt aaa 480Lys Val
Thr Asp Ala Ile Val Leu Leu Asp Arg Glu Gln Gly Gly Lys 145 150
155 160 gat aaa ctt caa gct cat ggt att cgt ctt cat tct gtt tgt act
ctt 528Asp Lys Leu Gln Ala His Gly Ile Arg Leu His Ser Val Cys Thr
Leu 165 170 175 tct aaa atg ctt gaa att ctt gaa caa caa aaa aaa gtt
gat gct gaa 576Ser Lys Met Leu Glu Ile Leu Glu Gln Gln Lys Lys Val
Asp Ala Glu 180 185 190 act gtt ggt cgt gtt aaa cgt ttt att caa gaa
aat gtt ttt gtt gct 624Thr Val Gly Arg Val Lys Arg Phe Ile Gln Glu
Asn Val Phe Val Ala 195 200 205 gct aat cat aat ggt tct cct ctt tct
att aaa gaa gct cct aaa gaa 672Ala Asn His Asn Gly Ser Pro Leu Ser
Ile Lys Glu Ala Pro Lys Glu 210 215 220 ctt tct ttt ggt gct cgt gct
gaa ctt cct cgt att cat cct gtt gct 720Leu Ser Phe Gly Ala Arg Ala
Glu Leu Pro Arg Ile His Pro Val Ala 225 230 235 240 tct aaa ctt ctt
cgt ctt atg caa aaa aaa gaa act aat ctt tgt ctt 768Ser Lys Leu Leu
Arg Leu Met Gln Lys Lys Glu Thr Asn Leu Cys Leu 245 250 255 tct gct
gat gtt tct ctt gct cgt gaa ctt ctt caa ctt gct gat gct 816Ser Ala
Asp Val Ser Leu Ala Arg Glu Leu Leu Gln Leu Ala Asp Ala 260 265 270
ctt ggt cct tct att tgt atg ctt aaa act cat gtt gat att ctt aat
864Leu Gly Pro Ser Ile Cys Met Leu Lys Thr His Val Asp Ile Leu Asn
275 280 285 gat ttt act ctt gat gtt atg aaa gaa ctt att act ctt gct
aaa tgt 912Asp Phe Thr Leu Asp Val Met Lys Glu Leu Ile Thr Leu Ala
Lys Cys 290 295 300 cat gaa ttt ctt att ttt gaa gat cgt aaa ttt gct
gat att ggt aat 960His Glu Phe Leu Ile Phe Glu Asp Arg Lys Phe Ala
Asp Ile Gly Asn 305 310 315 320 act gtt aaa aaa caa tat gaa ggt ggt
att ttt aaa att gct tct tgg 1008Thr Val Lys Lys Gln Tyr Glu Gly Gly
Ile Phe Lys Ile Ala Ser Trp 325 330 335 gct gat ctt gtt aat gct cat
gtt gtt cct ggt tct ggt gtt gtt aaa 1056Ala Asp Leu Val Asn Ala His
Val Val Pro Gly Ser Gly Val Val Lys 340 345 350 ggt ctt caa gaa gtt
ggt ctt cct ctt cat cgt ggt tgt ctt ctt att 1104Gly Leu Gln Glu Val
Gly Leu Pro Leu His Arg Gly Cys Leu Leu Ile 355 360 365 gct gaa atg
tct tct act ggt tct ctt gct act ggt gat tat act cgt 1152Ala Glu Met
Ser Ser Thr Gly Ser Leu Ala Thr Gly Asp Tyr Thr Arg 370 375 380 gct
gct gtt cgt atg gct gaa gaa cat tct gaa ttt gtt gtt ggt ttt 1200Ala
Ala Val Arg Met Ala Glu Glu His Ser Glu Phe Val Val Gly Phe 385 390
395 400 att tct ggt tct cgt gtt tct atg aaa cct gaa ttt ctt cat ctt
act 1248Ile Ser Gly Ser Arg Val Ser Met Lys Pro Glu Phe Leu His Leu
Thr 405 410 415 cct ggt gtt caa ctt gaa gct ggt ggt gat aat ctt ggt
caa caa tat 1296Pro Gly Val Gln Leu Glu Ala Gly Gly Asp Asn Leu Gly
Gln Gln Tyr 420 425 430 aat tct cct caa gaa gtt att ggt aaa cgt ggt
tct gat att att att 1344Asn Ser Pro Gln Glu Val Ile Gly Lys Arg Gly
Ser Asp Ile Ile Ile 435 440 445 gtt ggt cgt ggt att att tct gct gct
gat cgt ctt gaa gct gct gaa 1392Val Gly Arg Gly Ile Ile Ser Ala Ala
Asp Arg Leu Glu Ala Ala Glu 450 455 460 atg tat cgt aaa gct gct tgg
gaa gct tat ctt tct cgt ctt ggt gtt 1440Met Tyr Arg Lys Ala Ala Trp
Glu Ala Tyr Leu Ser Arg Leu Gly Val 465 470 475 480 taa
144310480PRThomo sapiens 10Met Ala Val Ala Arg Ala Ala Leu Gly Pro
Leu Val Thr Gly Leu Tyr 1 5 10 15 Asp Val Gln Ala Phe Lys Phe Gly
Asp Phe Val Leu Lys Ser Gly Leu 20 25 30 Ser Ser Pro Ile Tyr Ile
Asp Leu Arg Gly Ile Val Ser Arg Pro Arg 35 40 45 Leu Leu Ser Gln
Val Ala Asp Ile Leu Phe Gln Thr Ala Gln Asn Ala 50 55 60 Gly Ile
Ser Phe Asp Thr Val Cys Gly Val Pro Tyr Thr Ala Leu Pro 65 70 75 80
Leu Ala Thr Val Ile Cys Ser Thr Asn Gln Ile Pro Met Leu Ile Arg 85
90 95 Arg Lys Glu Thr Lys Asp Tyr Gly Thr Lys Arg Leu Val Glu Gly
Thr 100 105 110 Ile Asn Pro Gly Glu Thr Cys Leu Ile Ile Glu Asp Val
Val Thr Ser 115 120 125 Gly Ser Ser Val Leu Glu Thr Val Glu Val Leu
Gln Lys Glu Gly Leu 130 135 140 Lys Val Thr Asp Ala Ile Val Leu Leu
Asp Arg Glu Gln Gly Gly Lys 145 150 155 160 Asp Lys Leu Gln Ala His
Gly Ile Arg Leu His Ser Val Cys Thr Leu 165 170 175 Ser Lys Met Leu
Glu Ile Leu Glu Gln Gln Lys Lys Val Asp Ala Glu 180 185 190 Thr Val
Gly Arg Val Lys Arg Phe Ile Gln Glu Asn Val Phe Val Ala 195 200 205
Ala Asn His Asn Gly Ser Pro Leu Ser Ile Lys Glu Ala Pro Lys Glu 210
215 220 Leu Ser Phe Gly Ala Arg Ala Glu Leu Pro Arg Ile His Pro Val
Ala 225 230 235 240 Ser Lys Leu Leu Arg Leu Met Gln Lys Lys Glu Thr
Asn Leu Cys Leu 245 250 255 Ser Ala Asp Val Ser Leu Ala Arg Glu Leu
Leu Gln Leu Ala Asp Ala 260 265 270 Leu Gly Pro Ser Ile Cys Met Leu
Lys Thr His Val Asp Ile Leu Asn 275 280 285 Asp Phe Thr Leu Asp Val
Met Lys Glu Leu Ile Thr Leu Ala Lys Cys 290 295 300 His Glu Phe Leu
Ile Phe Glu Asp Arg Lys Phe Ala Asp Ile Gly Asn 305 310 315 320 Thr
Val Lys Lys Gln Tyr Glu Gly Gly Ile Phe Lys Ile Ala Ser Trp 325 330
335 Ala Asp Leu Val Asn Ala His Val Val Pro Gly Ser Gly Val Val Lys
340 345 350 Gly Leu Gln Glu Val Gly Leu Pro Leu His Arg Gly Cys Leu
Leu Ile 355 360 365 Ala Glu Met Ser Ser Thr Gly Ser Leu Ala Thr Gly
Asp Tyr Thr Arg 370 375 380 Ala Ala Val Arg Met Ala Glu Glu His Ser
Glu Phe Val Val Gly Phe 385 390 395 400 Ile Ser Gly Ser Arg Val Ser
Met Lys Pro Glu Phe Leu His Leu Thr 405 410 415 Pro Gly Val Gln Leu
Glu Ala Gly Gly Asp Asn Leu Gly Gln Gln Tyr 420 425 430 Asn Ser Pro
Gln Glu Val Ile Gly Lys Arg Gly Ser Asp Ile Ile Ile 435 440 445 Val
Gly Arg Gly Ile Ile Ser Ala Ala Asp Arg Leu Glu Ala Ala Glu 450 455
460 Met Tyr Arg Lys Ala Ala Trp Glu Ala Tyr Leu Ser Arg Leu Gly Val
465 470 475 480 111227DNAArtificial SequenceFusion construct
CDopt-UPRT 11atg gtg acc ggc ggc atg gcc tcc aag tgg gat caa aag
ggc atg gat 48Met Val Thr Gly Gly Met Ala Ser Lys Trp Asp Gln Lys
Gly Met Asp 1 5 10 15 atc gct tac gag gag gcc ctg ctg ggc tac aag
gag ggc ggc gtg cct 96Ile Ala Tyr Glu Glu Ala Leu Leu Gly Tyr Lys
Glu Gly Gly Val Pro 20 25 30 atc ggc ggc tgt ctg atc aac aac aag
gac ggc agt gtg ctg ggc agg 144Ile Gly Gly Cys Leu Ile Asn Asn Lys
Asp Gly Ser Val Leu Gly Arg 35 40 45 ggc cac aac atg agg ttc cag
aag ggc tcc gcc acc ctg cac ggc gag 192Gly His Asn Met Arg Phe Gln
Lys Gly Ser Ala Thr Leu His Gly Glu 50 55 60 atc tcc acc ctg gag
aac tgt ggc agg ctg gag ggc aag gtg tac aag 240Ile Ser Thr Leu Glu
Asn Cys Gly Arg Leu Glu Gly Lys Val Tyr Lys 65 70 75 80 gac acc acc
ctg tac acc acc ctg tcc cct tgt gac atg tgt acc ggc 288Asp Thr Thr
Leu Tyr Thr Thr Leu Ser Pro Cys Asp Met Cys Thr Gly 85 90 95 gct
atc atc atg tac ggc atc cct agg tgt gtg atc ggc gag aac gtg 336Ala
Ile Ile Met Tyr Gly Ile Pro Arg Cys Val Ile Gly Glu Asn Val 100 105
110 aac ttc aag tcc aag ggc gag aag tac ctg caa acc agg ggc cac gag
384Asn Phe Lys Ser Lys Gly Glu Lys Tyr Leu Gln Thr Arg Gly His Glu
115 120 125 gtg gtg gtt gtt gac gat gag agg tgt aag aag ctg atg aag
cag ttc 432Val Val Val Val Asp Asp Glu Arg Cys Lys Lys Leu Met Lys
Gln Phe 130 135 140 atc gac gag agg cct cag gac tgg ttc gag gat atc
ggc gag aac ccg 480Ile Asp Glu Arg Pro Gln Asp Trp Phe Glu Asp Ile
Gly Glu Asn Pro 145 150 155 160 tta ttc ttt ttg gct tct cca ttc ttg
tac ctt aca tat ctt ata tat 528Leu Phe Phe Leu Ala Ser Pro Phe Leu
Tyr Leu Thr Tyr Leu Ile Tyr 165 170 175 tat cca aac aaa ggg tct ttc
gtt agc aaa cct aga aat ctg caa aaa 576Tyr Pro Asn Lys Gly Ser Phe
Val Ser Lys Pro Arg Asn Leu Gln Lys 180 185 190 atg tct tcg gaa cca
ttt aag aac gtc tac ttg cta cct caa aca aac 624Met Ser Ser Glu Pro
Phe Lys Asn Val Tyr Leu Leu Pro Gln Thr Asn 195 200 205 caa ttg ctg
ggt ttg tac acc atc atc aga aat aag aat aca act aga 672Gln Leu Leu
Gly Leu Tyr Thr Ile Ile Arg Asn Lys Asn Thr Thr Arg 210 215 220 cct
gat ttc att ttc tac tcc gat aga atc atc aga ttg ttg gtt gaa 720Pro
Asp Phe Ile Phe Tyr Ser Asp Arg Ile Ile Arg Leu Leu Val Glu 225 230
235 240 gaa ggt ttg aac cat cta cct gtg caa aag caa att gtg gaa act
gac 768Glu Gly Leu Asn His Leu Pro Val Gln Lys Gln Ile Val Glu Thr
Asp 245 250 255 acc aac gaa aac ttc gaa ggt gtc tca ttc atg ggt aaa
atc tgt ggt 816Thr Asn Glu Asn Phe Glu Gly Val Ser Phe Met Gly Lys
Ile Cys Gly 260 265 270 gtt tcc att gtc aga gct ggt gaa tcg atg gag
caa gga tta aga gac 864Val Ser Ile Val Arg Ala Gly Glu Ser Met Glu
Gln Gly Leu Arg Asp 275 280 285 tgt tgt agg tct gtg cgt atc ggt aaa
att tta att caa agg gac gag 912Cys Cys Arg Ser Val Arg Ile Gly Lys
Ile Leu Ile Gln Arg Asp Glu 290 295 300 gag act gct tta cca aag tta
ttc tac gaa aaa tta cca gag gat ata 960Glu Thr Ala Leu Pro Lys Leu
Phe Tyr Glu Lys Leu Pro Glu Asp Ile 305 310 315 320 tct gaa agg tat
gtc ttc cta tta gac cca atg ctg gcc acc ggt ggt 1008Ser Glu Arg Tyr
Val Phe Leu Leu Asp Pro Met Leu Ala Thr Gly Gly 325 330 335 agt gct
atc atg gct aca gaa gtc ttg att aag aga ggt gtt aag cca 1056Ser Ala
Ile Met Ala Thr Glu Val Leu Ile Lys Arg Gly Val Lys Pro 340 345 350
gag aga att tac ttc tta aac cta atc tgt agt aag gaa ggg att gaa
1104Glu Arg Ile Tyr Phe Leu Asn Leu Ile Cys Ser Lys Glu Gly Ile Glu
355 360 365 aaa tac cat gcc gcc ttc cca gag gtc aga att gtt act ggt
gcc ctc 1152Lys Tyr His Ala Ala Phe Pro Glu Val Arg Ile Val Thr Gly
Ala Leu 370 375 380 gac aga ggt cta gat gaa aac aag tat cta gtt cca
ggg ttg ggt gac 1200Asp Arg Gly Leu Asp Glu Asn Lys Tyr Leu Val Pro
Gly Leu Gly Asp 385 390 395 400 ttt ggt gac aga tac tac tgt gtt taa
1227Phe Gly Asp Arg Tyr Tyr Cys Val 405 12408PRTArtificial
SequenceSynthetic Construct 12Met Val Thr Gly Gly Met Ala Ser Lys
Trp Asp Gln Lys Gly Met Asp 1 5 10 15 Ile Ala Tyr Glu Glu Ala Leu
Leu Gly Tyr Lys Glu Gly Gly Val Pro 20 25 30 Ile Gly Gly Cys Leu
Ile Asn Asn Lys Asp Gly Ser Val Leu Gly Arg 35 40 45 Gly His Asn
Met Arg Phe Gln Lys Gly Ser Ala Thr Leu His Gly Glu 50 55 60 Ile
Ser Thr Leu Glu Asn Cys Gly Arg Leu Glu Gly Lys Val Tyr Lys 65 70
75 80 Asp Thr Thr Leu Tyr Thr Thr Leu Ser Pro Cys Asp Met Cys Thr
Gly 85 90 95 Ala Ile Ile Met Tyr Gly Ile Pro Arg Cys Val Ile Gly
Glu Asn Val 100 105 110 Asn Phe Lys Ser Lys Gly Glu Lys Tyr Leu Gln
Thr Arg Gly His Glu 115 120 125 Val Val Val Val Asp Asp Glu Arg Cys
Lys Lys Leu Met Lys Gln Phe 130 135 140 Ile Asp Glu Arg Pro Gln Asp
Trp Phe Glu Asp Ile Gly Glu Asn Pro 145 150 155 160 Leu Phe Phe Leu
Ala Ser Pro Phe Leu Tyr Leu Thr Tyr Leu Ile Tyr 165 170 175 Tyr Pro
Asn Lys Gly Ser Phe Val Ser Lys Pro Arg Asn Leu Gln Lys 180 185 190
Met Ser Ser Glu Pro Phe Lys Asn Val Tyr Leu Leu Pro Gln Thr Asn 195
200 205 Gln Leu Leu Gly Leu Tyr Thr Ile Ile Arg Asn Lys Asn Thr Thr
Arg 210 215 220 Pro Asp Phe Ile Phe Tyr Ser Asp Arg Ile Ile Arg Leu
Leu Val Glu 225 230 235 240 Glu Gly Leu Asn His Leu Pro Val Gln Lys
Gln Ile Val Glu Thr Asp 245 250 255 Thr Asn Glu Asn Phe Glu Gly Val
Ser Phe Met Gly Lys Ile Cys Gly 260 265 270 Val Ser Ile Val Arg Ala
Gly Glu Ser Met Glu Gln Gly Leu Arg Asp 275 280 285 Cys Cys Arg Ser
Val Arg Ile Gly Lys Ile Leu Ile Gln Arg Asp Glu 290 295 300 Glu Thr
Ala Leu Pro Lys Leu Phe Tyr Glu Lys Leu Pro Glu Asp Ile 305 310 315
320 Ser Glu Arg Tyr Val Phe Leu Leu Asp Pro Met Leu Ala Thr Gly Gly
325 330 335 Ser Ala Ile Met Ala Thr Glu Val Leu Ile Lys Arg Gly Val
Lys Pro 340 345 350 Glu Arg Ile Tyr Phe Leu Asn Leu Ile Cys Ser Lys
Glu Gly Ile Glu 355 360 365 Lys Tyr His Ala Ala Phe Pro Glu Val Arg
Ile Val Thr Gly Ala Leu 370 375 380 Asp Arg Gly Leu Asp Glu Asn Lys
Tyr Leu Val Pro Gly Leu Gly Asp 385 390 395 400 Phe Gly Asp Arg Tyr
Tyr Cys Val 405 131287DNAArtificial SequenceFusion construction -
CDopt - linker - UPRT 13atg gtg acc ggc ggc atg gcc tcc aag tgg gat
caa aag ggc atg gat 48Met Val Thr Gly Gly Met Ala Ser Lys Trp Asp
Gln Lys Gly Met Asp 1 5 10 15 atc gct tac gag gag gcc ctg ctg ggc
tac aag gag ggc ggc gtg cct 96Ile Ala Tyr Glu Glu Ala Leu Leu Gly
Tyr Lys Glu Gly Gly Val Pro 20 25 30 atc ggc ggc tgt ctg atc aac
aac aag gac ggc agt gtg ctg ggc agg 144Ile Gly Gly Cys Leu Ile Asn
Asn Lys Asp Gly Ser Val Leu Gly Arg 35 40 45 ggc cac aac atg agg
ttc cag aag ggc tcc gcc acc ctg cac ggc gag 192Gly His Asn Met Arg
Phe Gln Lys Gly Ser Ala Thr Leu His Gly Glu 50 55 60 atc tcc acc
ctg gag aac tgt
ggc agg ctg gag ggc aag gtg tac aag 240Ile Ser Thr Leu Glu Asn Cys
Gly Arg Leu Glu Gly Lys Val Tyr Lys 65 70 75 80 gac acc acc ctg tac
acc acc ctg tcc cct tgt gac atg tgt acc ggc 288Asp Thr Thr Leu Tyr
Thr Thr Leu Ser Pro Cys Asp Met Cys Thr Gly 85 90 95 gct atc atc
atg tac ggc atc cct agg tgt gtg atc ggc gag aac gtg 336Ala Ile Ile
Met Tyr Gly Ile Pro Arg Cys Val Ile Gly Glu Asn Val 100 105 110 aac
ttc aag tcc aag ggc gag aag tac ctg caa acc agg ggc cac gag 384Asn
Phe Lys Ser Lys Gly Glu Lys Tyr Leu Gln Thr Arg Gly His Glu 115 120
125 gtg gtg gtt gtt gac gat gag agg tgt aag aag ctg atg aag cag ttc
432Val Val Val Val Asp Asp Glu Arg Cys Lys Lys Leu Met Lys Gln Phe
130 135 140 atc gac gag agg cct cag gac tgg ttc gag gat atc ggc gag
tcc ggc 480Ile Asp Glu Arg Pro Gln Asp Trp Phe Glu Asp Ile Gly Glu
Ser Gly 145 150 155 160 ggc ggc gcc tcc ggc ggc ggc gcc tcc ggc ggc
ggc gcc tcc ggc ggc 528Gly Gly Ala Ser Gly Gly Gly Ala Ser Gly Gly
Gly Ala Ser Gly Gly 165 170 175 ggc gcc aac ccg tta ttc ttt ttg gct
tct cca ttc ttg tac ctt aca 576Gly Ala Asn Pro Leu Phe Phe Leu Ala
Ser Pro Phe Leu Tyr Leu Thr 180 185 190 tat ctt ata tat tat cca aac
aaa ggg tct ttc gtt agc aaa cct aga 624Tyr Leu Ile Tyr Tyr Pro Asn
Lys Gly Ser Phe Val Ser Lys Pro Arg 195 200 205 aat ctg caa aaa atg
tct tcg gaa cca ttt aag aac gtc tac ttg cta 672Asn Leu Gln Lys Met
Ser Ser Glu Pro Phe Lys Asn Val Tyr Leu Leu 210 215 220 cct caa aca
aac caa ttg ctg ggt ttg tac acc atc atc aga aat aag 720Pro Gln Thr
Asn Gln Leu Leu Gly Leu Tyr Thr Ile Ile Arg Asn Lys 225 230 235 240
aat aca act aga cct gat ttc att ttc tac tcc gat aga atc atc aga
768Asn Thr Thr Arg Pro Asp Phe Ile Phe Tyr Ser Asp Arg Ile Ile Arg
245 250 255 ttg ttg gtt gaa gaa ggt ttg aac cat cta cct gtg caa aag
caa att 816Leu Leu Val Glu Glu Gly Leu Asn His Leu Pro Val Gln Lys
Gln Ile 260 265 270 gtg gaa act gac acc aac gaa aac ttc gaa ggt gtc
tca ttc atg ggt 864Val Glu Thr Asp Thr Asn Glu Asn Phe Glu Gly Val
Ser Phe Met Gly 275 280 285 aaa atc tgt ggt gtt tcc att gtc aga gct
ggt gaa tcg atg gag caa 912Lys Ile Cys Gly Val Ser Ile Val Arg Ala
Gly Glu Ser Met Glu Gln 290 295 300 gga tta aga gac tgt tgt agg tct
gtg cgt atc ggt aaa att tta att 960Gly Leu Arg Asp Cys Cys Arg Ser
Val Arg Ile Gly Lys Ile Leu Ile 305 310 315 320 caa agg gac gag gag
act gct tta cca aag tta ttc tac gaa aaa tta 1008Gln Arg Asp Glu Glu
Thr Ala Leu Pro Lys Leu Phe Tyr Glu Lys Leu 325 330 335 cca gag gat
ata tct gaa agg tat gtc ttc cta tta gac cca atg ctg 1056Pro Glu Asp
Ile Ser Glu Arg Tyr Val Phe Leu Leu Asp Pro Met Leu 340 345 350 gcc
acc ggt ggt agt gct atc atg gct aca gaa gtc ttg att aag aga 1104Ala
Thr Gly Gly Ser Ala Ile Met Ala Thr Glu Val Leu Ile Lys Arg 355 360
365 ggt gtt aag cca gag aga att tac ttc tta aac cta atc tgt agt aag
1152Gly Val Lys Pro Glu Arg Ile Tyr Phe Leu Asn Leu Ile Cys Ser Lys
370 375 380 gaa ggg att gaa aaa tac cat gcc gcc ttc cca gag gtc aga
att gtt 1200Glu Gly Ile Glu Lys Tyr His Ala Ala Phe Pro Glu Val Arg
Ile Val 385 390 395 400 act ggt gcc ctc gac aga ggt cta gat gaa aac
aag tat cta gtt cca 1248Thr Gly Ala Leu Asp Arg Gly Leu Asp Glu Asn
Lys Tyr Leu Val Pro 405 410 415 ggg ttg ggt gac ttt ggt gac aga tac
tac tgt gtt taa 1287Gly Leu Gly Asp Phe Gly Asp Arg Tyr Tyr Cys Val
420 425 14428PRTArtificial SequenceSynthetic Construct 14Met Val
Thr Gly Gly Met Ala Ser Lys Trp Asp Gln Lys Gly Met Asp 1 5 10 15
Ile Ala Tyr Glu Glu Ala Leu Leu Gly Tyr Lys Glu Gly Gly Val Pro 20
25 30 Ile Gly Gly Cys Leu Ile Asn Asn Lys Asp Gly Ser Val Leu Gly
Arg 35 40 45 Gly His Asn Met Arg Phe Gln Lys Gly Ser Ala Thr Leu
His Gly Glu 50 55 60 Ile Ser Thr Leu Glu Asn Cys Gly Arg Leu Glu
Gly Lys Val Tyr Lys 65 70 75 80 Asp Thr Thr Leu Tyr Thr Thr Leu Ser
Pro Cys Asp Met Cys Thr Gly 85 90 95 Ala Ile Ile Met Tyr Gly Ile
Pro Arg Cys Val Ile Gly Glu Asn Val 100 105 110 Asn Phe Lys Ser Lys
Gly Glu Lys Tyr Leu Gln Thr Arg Gly His Glu 115 120 125 Val Val Val
Val Asp Asp Glu Arg Cys Lys Lys Leu Met Lys Gln Phe 130 135 140 Ile
Asp Glu Arg Pro Gln Asp Trp Phe Glu Asp Ile Gly Glu Ser Gly 145 150
155 160 Gly Gly Ala Ser Gly Gly Gly Ala Ser Gly Gly Gly Ala Ser Gly
Gly 165 170 175 Gly Ala Asn Pro Leu Phe Phe Leu Ala Ser Pro Phe Leu
Tyr Leu Thr 180 185 190 Tyr Leu Ile Tyr Tyr Pro Asn Lys Gly Ser Phe
Val Ser Lys Pro Arg 195 200 205 Asn Leu Gln Lys Met Ser Ser Glu Pro
Phe Lys Asn Val Tyr Leu Leu 210 215 220 Pro Gln Thr Asn Gln Leu Leu
Gly Leu Tyr Thr Ile Ile Arg Asn Lys 225 230 235 240 Asn Thr Thr Arg
Pro Asp Phe Ile Phe Tyr Ser Asp Arg Ile Ile Arg 245 250 255 Leu Leu
Val Glu Glu Gly Leu Asn His Leu Pro Val Gln Lys Gln Ile 260 265 270
Val Glu Thr Asp Thr Asn Glu Asn Phe Glu Gly Val Ser Phe Met Gly 275
280 285 Lys Ile Cys Gly Val Ser Ile Val Arg Ala Gly Glu Ser Met Glu
Gln 290 295 300 Gly Leu Arg Asp Cys Cys Arg Ser Val Arg Ile Gly Lys
Ile Leu Ile 305 310 315 320 Gln Arg Asp Glu Glu Thr Ala Leu Pro Lys
Leu Phe Tyr Glu Lys Leu 325 330 335 Pro Glu Asp Ile Ser Glu Arg Tyr
Val Phe Leu Leu Asp Pro Met Leu 340 345 350 Ala Thr Gly Gly Ser Ala
Ile Met Ala Thr Glu Val Leu Ile Lys Arg 355 360 365 Gly Val Lys Pro
Glu Arg Ile Tyr Phe Leu Asn Leu Ile Cys Ser Lys 370 375 380 Glu Gly
Ile Glu Lys Tyr His Ala Ala Phe Pro Glu Val Arg Ile Val 385 390 395
400 Thr Gly Ala Leu Asp Arg Gly Leu Asp Glu Asn Lys Tyr Leu Val Pro
405 410 415 Gly Leu Gly Asp Phe Gly Asp Arg Tyr Tyr Cys Val 420 425
151200DNAArtificial SequenceFusion Construct - CDopt3 - OPRT 15atg
gtg acc ggc ggc atg gcc tcc aag tgg gat caa aag ggc atg gat 48Met
Val Thr Gly Gly Met Ala Ser Lys Trp Asp Gln Lys Gly Met Asp 1 5 10
15 atc gct tac gag gag gcc ctg ctg ggc tac aag gag ggc ggc gtg cct
96Ile Ala Tyr Glu Glu Ala Leu Leu Gly Tyr Lys Glu Gly Gly Val Pro
20 25 30 atc ggc ggc tgt ctg atc aac aac aag gac ggc agt gtg ctg
ggc agg 144Ile Gly Gly Cys Leu Ile Asn Asn Lys Asp Gly Ser Val Leu
Gly Arg 35 40 45 ggc cac aac atg agg ttc cag aag ggc tcc gcc acc
ctg cac ggc gag 192Gly His Asn Met Arg Phe Gln Lys Gly Ser Ala Thr
Leu His Gly Glu 50 55 60 atc tcc acc ctg gag aac tgt ggc agg ctg
gag ggc aag gtg tac aag 240Ile Ser Thr Leu Glu Asn Cys Gly Arg Leu
Glu Gly Lys Val Tyr Lys 65 70 75 80 gac acc acc ctg tac acc acc ctg
tcc cct tgt gac atg tgt acc ggc 288Asp Thr Thr Leu Tyr Thr Thr Leu
Ser Pro Cys Asp Met Cys Thr Gly 85 90 95 gct atc atc atg tac ggc
atc cct agg tgt gtg atc ggc gag aac gtg 336Ala Ile Ile Met Tyr Gly
Ile Pro Arg Cys Val Ile Gly Glu Asn Val 100 105 110 aac ttc aag tcc
aag ggc gag aag tac ctg caa acc agg ggc cac gag 384Asn Phe Lys Ser
Lys Gly Glu Lys Tyr Leu Gln Thr Arg Gly His Glu 115 120 125 gtg gtg
gtt gtt gac gat gag agg tgt aag aag ctg atg aag cag ttc 432Val Val
Val Val Asp Asp Glu Arg Cys Lys Lys Leu Met Lys Gln Phe 130 135 140
atc gac gag agg cct cag gac tgg ttc gag gat atc ggc gag gcg gtc
480Ile Asp Glu Arg Pro Gln Asp Trp Phe Glu Asp Ile Gly Glu Ala Val
145 150 155 160 gct cgt gca gct ttg ggg cca ttg gtg acg ggt ctg tac
gac gtg cag 528Ala Arg Ala Ala Leu Gly Pro Leu Val Thr Gly Leu Tyr
Asp Val Gln 165 170 175 gct ttc aag ttt ggg gac ttc gtg ctg aag agc
ggg ctt tcc tcc ccc 576Ala Phe Lys Phe Gly Asp Phe Val Leu Lys Ser
Gly Leu Ser Ser Pro 180 185 190 atc tac atc gat ctg cgg ggc atc gtg
tct cga ccg cgt ctt ctg agt 624Ile Tyr Ile Asp Leu Arg Gly Ile Val
Ser Arg Pro Arg Leu Leu Ser 195 200 205 cag gtt gca gat att tta ttc
caa act gcc caa aat gca ggc atc agt 672Gln Val Ala Asp Ile Leu Phe
Gln Thr Ala Gln Asn Ala Gly Ile Ser 210 215 220 ttt gac acc gtg tgt
gga gtg cct tat aca gct ttg cca ttg gct aca 720Phe Asp Thr Val Cys
Gly Val Pro Tyr Thr Ala Leu Pro Leu Ala Thr 225 230 235 240 gtt atc
tgt tca acc aat caa att cca atg ctt att aga agg aaa gaa 768Val Ile
Cys Ser Thr Asn Gln Ile Pro Met Leu Ile Arg Arg Lys Glu 245 250 255
aca aag gat tat gga act aag cgt ctt gta gaa gga act att aat cca
816Thr Lys Asp Tyr Gly Thr Lys Arg Leu Val Glu Gly Thr Ile Asn Pro
260 265 270 gga gaa acc tgt tta atc att gaa gat gtt gtc acc agt gga
tct agt 864Gly Glu Thr Cys Leu Ile Ile Glu Asp Val Val Thr Ser Gly
Ser Ser 275 280 285 gtt ttg gaa act gtt gag gtt ctt cag aag gag ggc
ttg aag gtc act 912Val Leu Glu Thr Val Glu Val Leu Gln Lys Glu Gly
Leu Lys Val Thr 290 295 300 gat gcc ata gtg ctg ttg gac aga gag cag
gga ggc aag gac aag ttg 960Asp Ala Ile Val Leu Leu Asp Arg Glu Gln
Gly Gly Lys Asp Lys Leu 305 310 315 320 cag gcg cac ggg atc cgc ctc
cac tca gtg tgt aca ttg tcc aaa atg 1008Gln Ala His Gly Ile Arg Leu
His Ser Val Cys Thr Leu Ser Lys Met 325 330 335 ctg gag att ctc gag
cag cag aaa aaa gtt gat gct gag aca gtt ggg 1056Leu Glu Ile Leu Glu
Gln Gln Lys Lys Val Asp Ala Glu Thr Val Gly 340 345 350 aga gtg aag
agg ttt att cag gag aat gtc ttt gtg gca gcg aat cat 1104Arg Val Lys
Arg Phe Ile Gln Glu Asn Val Phe Val Ala Ala Asn His 355 360 365 aat
ggt tct ccc ctt tct ata aag gaa gca ccc aaa gaa ctc agc ttc 1152Asn
Gly Ser Pro Leu Ser Ile Lys Glu Ala Pro Lys Glu Leu Ser Phe 370 375
380 ggt gca cgt gca gag ctg ccc agg atc cac cca gtt gca tcg aag taa
1200Gly Ala Arg Ala Glu Leu Pro Arg Ile His Pro Val Ala Ser Lys 385
390 395 16399PRTArtificial SequenceSynthetic Construct 16Met Val
Thr Gly Gly Met Ala Ser Lys Trp Asp Gln Lys Gly Met Asp 1 5 10 15
Ile Ala Tyr Glu Glu Ala Leu Leu Gly Tyr Lys Glu Gly Gly Val Pro 20
25 30 Ile Gly Gly Cys Leu Ile Asn Asn Lys Asp Gly Ser Val Leu Gly
Arg 35 40 45 Gly His Asn Met Arg Phe Gln Lys Gly Ser Ala Thr Leu
His Gly Glu 50 55 60 Ile Ser Thr Leu Glu Asn Cys Gly Arg Leu Glu
Gly Lys Val Tyr Lys 65 70 75 80 Asp Thr Thr Leu Tyr Thr Thr Leu Ser
Pro Cys Asp Met Cys Thr Gly 85 90 95 Ala Ile Ile Met Tyr Gly Ile
Pro Arg Cys Val Ile Gly Glu Asn Val 100 105 110 Asn Phe Lys Ser Lys
Gly Glu Lys Tyr Leu Gln Thr Arg Gly His Glu 115 120 125 Val Val Val
Val Asp Asp Glu Arg Cys Lys Lys Leu Met Lys Gln Phe 130 135 140 Ile
Asp Glu Arg Pro Gln Asp Trp Phe Glu Asp Ile Gly Glu Ala Val 145 150
155 160 Ala Arg Ala Ala Leu Gly Pro Leu Val Thr Gly Leu Tyr Asp Val
Gln 165 170 175 Ala Phe Lys Phe Gly Asp Phe Val Leu Lys Ser Gly Leu
Ser Ser Pro 180 185 190 Ile Tyr Ile Asp Leu Arg Gly Ile Val Ser Arg
Pro Arg Leu Leu Ser 195 200 205 Gln Val Ala Asp Ile Leu Phe Gln Thr
Ala Gln Asn Ala Gly Ile Ser 210 215 220 Phe Asp Thr Val Cys Gly Val
Pro Tyr Thr Ala Leu Pro Leu Ala Thr 225 230 235 240 Val Ile Cys Ser
Thr Asn Gln Ile Pro Met Leu Ile Arg Arg Lys Glu 245 250 255 Thr Lys
Asp Tyr Gly Thr Lys Arg Leu Val Glu Gly Thr Ile Asn Pro 260 265 270
Gly Glu Thr Cys Leu Ile Ile Glu Asp Val Val Thr Ser Gly Ser Ser 275
280 285 Val Leu Glu Thr Val Glu Val Leu Gln Lys Glu Gly Leu Lys Val
Thr 290 295 300 Asp Ala Ile Val Leu Leu Asp Arg Glu Gln Gly Gly Lys
Asp Lys Leu 305 310 315 320 Gln Ala His Gly Ile Arg Leu His Ser Val
Cys Thr Leu Ser Lys Met 325 330 335 Leu Glu Ile Leu Glu Gln Gln Lys
Lys Val Asp Ala Glu Thr Val Gly 340 345 350 Arg Val Lys Arg Phe Ile
Gln Glu Asn Val Phe Val Ala Ala Asn His 355 360 365 Asn Gly Ser Pro
Leu Ser Ile Lys Glu Ala Pro Lys Glu Leu Ser Phe 370 375 380 Gly Ala
Arg Ala Glu Leu Pro Arg Ile His Pro Val Ala Ser Lys 385 390 395
171260DNAArtificial SequenceFusion Construct - CDopt3 - linker -
OPRT 17atg gtg acc ggc ggc atg gcc tcc aag tgg gat caa aag ggc atg
gat 48Met Val Thr Gly Gly Met Ala Ser Lys Trp Asp Gln Lys Gly Met
Asp 1 5 10 15 atc gct tac gag gag gcc ctg ctg ggc tac aag gag ggc
ggc gtg cct 96Ile Ala Tyr Glu Glu Ala Leu Leu Gly Tyr Lys Glu Gly
Gly Val Pro 20 25 30 atc ggc ggc tgt ctg atc aac aac aag gac ggc
agt gtg ctg ggc agg 144Ile Gly Gly Cys Leu Ile Asn Asn Lys Asp Gly
Ser Val Leu Gly Arg 35 40 45 ggc cac aac atg agg ttc cag aag ggc
tcc gcc acc ctg cac ggc gag 192Gly His Asn Met Arg Phe Gln Lys Gly
Ser Ala Thr Leu His Gly Glu 50 55 60 atc tcc acc ctg gag aac tgt
ggc agg ctg gag ggc aag gtg tac aag 240Ile Ser Thr Leu Glu Asn Cys
Gly Arg Leu Glu Gly Lys Val Tyr Lys 65 70 75
80 gac acc acc ctg tac acc acc ctg tcc cct tgt gac atg tgt acc ggc
288Asp Thr Thr Leu Tyr Thr Thr Leu Ser Pro Cys Asp Met Cys Thr Gly
85 90 95 gct atc atc atg tac ggc atc cct agg tgt gtg atc ggc gag
aac gtg 336Ala Ile Ile Met Tyr Gly Ile Pro Arg Cys Val Ile Gly Glu
Asn Val 100 105 110 aac ttc aag tcc aag ggc gag aag tac ctg caa acc
agg ggc cac gag 384Asn Phe Lys Ser Lys Gly Glu Lys Tyr Leu Gln Thr
Arg Gly His Glu 115 120 125 gtg gtg gtt gtt gac gat gag agg tgt aag
aag ctg atg aag cag ttc 432Val Val Val Val Asp Asp Glu Arg Cys Lys
Lys Leu Met Lys Gln Phe 130 135 140 atc gac gag agg cct cag gac tgg
ttc gag gat atc ggc gag tcc ggc 480Ile Asp Glu Arg Pro Gln Asp Trp
Phe Glu Asp Ile Gly Glu Ser Gly 145 150 155 160 ggc ggc gcc tcc ggc
ggc ggc gcc tcc ggc ggc ggc gcc tcc ggc ggc 528Gly Gly Ala Ser Gly
Gly Gly Ala Ser Gly Gly Gly Ala Ser Gly Gly 165 170 175 ggc gcc gcg
gtc gct cgt gca gct ttg ggg cca ttg gtg acg ggt ctg 576Gly Ala Ala
Val Ala Arg Ala Ala Leu Gly Pro Leu Val Thr Gly Leu 180 185 190 tac
gac gtg cag gct ttc aag ttt ggg gac ttc gtg ctg aag agc ggg 624Tyr
Asp Val Gln Ala Phe Lys Phe Gly Asp Phe Val Leu Lys Ser Gly 195 200
205 ctt tcc tcc ccc atc tac atc gat ctg cgg ggc atc gtg tct cga ccg
672Leu Ser Ser Pro Ile Tyr Ile Asp Leu Arg Gly Ile Val Ser Arg Pro
210 215 220 cgt ctt ctg agt cag gtt gca gat att tta ttc caa act gcc
caa aat 720Arg Leu Leu Ser Gln Val Ala Asp Ile Leu Phe Gln Thr Ala
Gln Asn 225 230 235 240 gca ggc atc agt ttt gac acc gtg tgt gga gtg
cct tat aca gct ttg 768Ala Gly Ile Ser Phe Asp Thr Val Cys Gly Val
Pro Tyr Thr Ala Leu 245 250 255 cca ttg gct aca gtt atc tgt tca acc
aat caa att cca atg ctt att 816Pro Leu Ala Thr Val Ile Cys Ser Thr
Asn Gln Ile Pro Met Leu Ile 260 265 270 aga agg aaa gaa aca aag gat
tat gga act aag cgt ctt gta gaa gga 864Arg Arg Lys Glu Thr Lys Asp
Tyr Gly Thr Lys Arg Leu Val Glu Gly 275 280 285 act att aat cca gga
gaa acc tgt tta atc att gaa gat gtt gtc acc 912Thr Ile Asn Pro Gly
Glu Thr Cys Leu Ile Ile Glu Asp Val Val Thr 290 295 300 agt gga tct
agt gtt ttg gaa act gtt gag gtt ctt cag aag gag ggc 960Ser Gly Ser
Ser Val Leu Glu Thr Val Glu Val Leu Gln Lys Glu Gly 305 310 315 320
ttg aag gtc act gat gcc ata gtg ctg ttg gac aga gag cag gga ggc
1008Leu Lys Val Thr Asp Ala Ile Val Leu Leu Asp Arg Glu Gln Gly Gly
325 330 335 aag gac aag ttg cag gcg cac ggg atc cgc ctc cac tca gtg
tgt aca 1056Lys Asp Lys Leu Gln Ala His Gly Ile Arg Leu His Ser Val
Cys Thr 340 345 350 ttg tcc aaa atg ctg gag att ctc gag cag cag aaa
aaa gtt gat gct 1104Leu Ser Lys Met Leu Glu Ile Leu Glu Gln Gln Lys
Lys Val Asp Ala 355 360 365 gag aca gtt ggg aga gtg aag agg ttt att
cag gag aat gtc ttt gtg 1152Glu Thr Val Gly Arg Val Lys Arg Phe Ile
Gln Glu Asn Val Phe Val 370 375 380 gca gcg aat cat aat ggt tct ccc
ctt tct ata aag gaa gca ccc aaa 1200Ala Ala Asn His Asn Gly Ser Pro
Leu Ser Ile Lys Glu Ala Pro Lys 385 390 395 400 gaa ctc agc ttc ggt
gca cgt gca gag ctg ccc agg atc cac cca gtt 1248Glu Leu Ser Phe Gly
Ala Arg Ala Glu Leu Pro Arg Ile His Pro Val 405 410 415 gca tcg aag
taa 1260Ala Ser Lys 18419PRTArtificial SequenceSynthetic Construct
18Met Val Thr Gly Gly Met Ala Ser Lys Trp Asp Gln Lys Gly Met Asp 1
5 10 15 Ile Ala Tyr Glu Glu Ala Leu Leu Gly Tyr Lys Glu Gly Gly Val
Pro 20 25 30 Ile Gly Gly Cys Leu Ile Asn Asn Lys Asp Gly Ser Val
Leu Gly Arg 35 40 45 Gly His Asn Met Arg Phe Gln Lys Gly Ser Ala
Thr Leu His Gly Glu 50 55 60 Ile Ser Thr Leu Glu Asn Cys Gly Arg
Leu Glu Gly Lys Val Tyr Lys 65 70 75 80 Asp Thr Thr Leu Tyr Thr Thr
Leu Ser Pro Cys Asp Met Cys Thr Gly 85 90 95 Ala Ile Ile Met Tyr
Gly Ile Pro Arg Cys Val Ile Gly Glu Asn Val 100 105 110 Asn Phe Lys
Ser Lys Gly Glu Lys Tyr Leu Gln Thr Arg Gly His Glu 115 120 125 Val
Val Val Val Asp Asp Glu Arg Cys Lys Lys Leu Met Lys Gln Phe 130 135
140 Ile Asp Glu Arg Pro Gln Asp Trp Phe Glu Asp Ile Gly Glu Ser Gly
145 150 155 160 Gly Gly Ala Ser Gly Gly Gly Ala Ser Gly Gly Gly Ala
Ser Gly Gly 165 170 175 Gly Ala Ala Val Ala Arg Ala Ala Leu Gly Pro
Leu Val Thr Gly Leu 180 185 190 Tyr Asp Val Gln Ala Phe Lys Phe Gly
Asp Phe Val Leu Lys Ser Gly 195 200 205 Leu Ser Ser Pro Ile Tyr Ile
Asp Leu Arg Gly Ile Val Ser Arg Pro 210 215 220 Arg Leu Leu Ser Gln
Val Ala Asp Ile Leu Phe Gln Thr Ala Gln Asn 225 230 235 240 Ala Gly
Ile Ser Phe Asp Thr Val Cys Gly Val Pro Tyr Thr Ala Leu 245 250 255
Pro Leu Ala Thr Val Ile Cys Ser Thr Asn Gln Ile Pro Met Leu Ile 260
265 270 Arg Arg Lys Glu Thr Lys Asp Tyr Gly Thr Lys Arg Leu Val Glu
Gly 275 280 285 Thr Ile Asn Pro Gly Glu Thr Cys Leu Ile Ile Glu Asp
Val Val Thr 290 295 300 Ser Gly Ser Ser Val Leu Glu Thr Val Glu Val
Leu Gln Lys Glu Gly 305 310 315 320 Leu Lys Val Thr Asp Ala Ile Val
Leu Leu Asp Arg Glu Gln Gly Gly 325 330 335 Lys Asp Lys Leu Gln Ala
His Gly Ile Arg Leu His Ser Val Cys Thr 340 345 350 Leu Ser Lys Met
Leu Glu Ile Leu Glu Gln Gln Lys Lys Val Asp Ala 355 360 365 Glu Thr
Val Gly Arg Val Lys Arg Phe Ile Gln Glu Asn Val Phe Val 370 375 380
Ala Ala Asn His Asn Gly Ser Pro Leu Ser Ile Lys Glu Ala Pro Lys 385
390 395 400 Glu Leu Ser Phe Gly Ala Arg Ala Glu Leu Pro Arg Ile His
Pro Val 405 410 415 Ala Ser Lys 1911892DNAArtificial SequenceRCR
Vector - pAC3-yCD2 19tagttattaa tagtaatcaa ttacggggtc attagttcat
agcccatata tggagttccg 60cgttacataa cttacggtaa atggcccgcc tggctgaccg
cccaacgacc cccgcccatt 120gacgtcaata atgacgtatg ttcccatagt
aacgccaata gggactttcc attgacgtca 180atgggtggag tatttacggt
aaactgccca cttggcagta catcaagtgt atcatatgcc 240aagtacgccc
cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
300catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
tcgctattac 360catggtgatg cggttttggc agtacatcaa tgggcgtgga
tagcggtttg actcacgggg 420atttccaagt ctccacccca ttgacgtcaa
tgggagtttg ttttggcacc aaaatcaacg 480ggactttcca aaatgtcgta
acaactccgc cccattgacg caaatgggcg gtaggcgtgt 540acggtgggag
gtctatataa gcagagctgg tttagtgaac cggcgccagt cctccgattg
600actgagtcgc ccgggtaccc gtgtatccaa taaaccctct tgcagttgca
tccgacttgt 660ggtctcgctg ttccttggga gggtctcctc tgagtgattg
actacccgtc agcgggggtc 720tttcatttgg gggctcgtcc gggatcggga
gacccctgcc cagggaccac cgacccacca 780ccgggaggta agctggccag
caacttatct gtgtctgtcc gattgtctag tgtctatgac 840tgattttatg
cgcctgcgtc ggtactagtt agctaactag ctctgtatct ggcggacccg
900tggtggaact gacgagttcg gaacacccgg ccgcaaccct gggagacgtc
ccagggactt 960cgggggccgt ttttgtggcc cgacctgagt ccaaaaatcc
cgatcgtttt ggactctttg 1020gtgcaccccc cttagaggag ggatatgtgg
ttctggtagg agacgagaac ctaaaacagt 1080tcccgcctcc gtctgaattt
ttgctttcgg tttgggaccg aagccgcgcc gcgcgtcttg 1140tctgctgcag
catcgttctg tgttgtctct gtctgactgt gtttctgtat ttgtctgaga
1200atatgggcca gactgttacc actcccttaa gtttgacctt aggtcactgg
aaagatgtcg 1260agcggatcgc tcacaaccag tcggtagatg tcaagaagag
acgttgggtt accttctgct 1320ctgcagaatg gccaaccttt aacgtcggat
ggccgcgaga cggcaccttt aaccgagacc 1380tcatcaccca ggttaagatc
aaggtctttt cacctggccc gcatggacac ccagaccagg 1440tcccctacat
cgtgacctgg gaagccttgg cttttgaccc ccctccctgg gtcaagccct
1500ttgtacaccc taagcctccg cctcctcttc ctccatccgc cccgtctctc
ccccttgaac 1560ctcctcgttc gaccccgcct cgatcctccc tttatccagc
cctcactcct tctctaggcg 1620ccaaacctaa acctcaagtt ctttctgaca
gtggggggcc gctcatcgac ctacttacag 1680aagacccccc gccttatagg
gacccaagac cacccccttc cgacagggac ggaaatggtg 1740gagaagcgac
ccctgcggga gaggcaccgg acccctcccc aatggcatct cgcctacgtg
1800ggagacggga gccccctgtg gccgactcca ctacctcgca ggcattcccc
ctccgcgcag 1860gaggaaacgg acagcttcaa tactggccgt tctcctcttc
tgacctttac aactggaaaa 1920ataataaccc ttctttttct gaagatccag
gtaaactgac agctctgatc gagtctgttc 1980tcatcaccca tcagcccacc
tgggacgact gtcagcagct gttggggact ctgctgaccg 2040gagaagaaaa
acaacgggtg ctcttagagg ctagaaaggc ggtgcggggc gatgatgggc
2100gccccactca actgcccaat gaagtcgatg ccgcttttcc cctcgagcgc
ccagactggg 2160attacaccac ccaggcaggt aggaaccacc tagtccacta
tcgccagttg ctcctagcgg 2220gtctccaaaa cgcgggcaga agccccacca
atttggccaa ggtaaaagga ataacacaag 2280ggcccaatga gtctccctcg
gccttcctag agagacttaa ggaagcctat cgcaggtaca 2340ctccttatga
ccctgaggac ccagggcaag aaactaatgt gtctatgtct ttcatttggc
2400agtctgcccc agacattggg agaaagttag agaggttaga agatttaaaa
aacaagacgc 2460ttggagattt ggttagagag gcagaaaaga tctttaataa
acgagaaacc ccggaagaaa 2520gagaggaacg tatcaggaga gaaacagagg
aaaaagaaga acgccgtagg acagaggatg 2580agcagaaaga gaaagaaaga
gatcgtagga gacatagaga gatgagcaag ctattggcca 2640ctgtcgttag
tggacagaaa caggatagac agggaggaga acgaaggagg tcccaactcg
2700atcgcgacca gtgtgcctac tgcaaagaaa aggggcactg ggctaaagat
tgtcccaaga 2760aaccacgagg acctcgggga ccaagacccc agacctccct
cctgacccta gatgactagg 2820gaggtcaggg tcaggagccc ccccctgaac
ccaggataac cctcaaagtc ggggggcaac 2880ccgtcacctt cctggtagat
actggggccc aacactccgt gctgacccaa aatcctggac 2940ccctaagtga
taagtctgcc tgggtccaag gggctactgg aggaaagcgg tatcgctgga
3000ccacggatcg caaagtacat ctagctaccg gtaaggtcac ccactctttc
ctccatgtac 3060cagactgtcc ctatcctctg ttaggaagag atttgctgac
taaactaaaa gcccaaatcc 3120actttgaggg atcaggagcc caggttatgg
gaccaatggg gcagcccctg caagtgttga 3180ccctaaatat agaagatgag
catcggctac atgagacctc aaaagagcca gatgtttctc 3240tagggtccac
atggctgtct gattttcctc aggcctgggc ggaaaccggg ggcatgggac
3300tggcagttcg ccaagctcct ctgatcatac ctctgaaagc aacctctacc
cccgtgtcca 3360taaaacaata ccccatgtca caagaagcca gactggggat
caagccccac atacagagac 3420tgttggacca gggaatactg gtaccctgcc
agtccccctg gaacacgccc ctgctacccg 3480ttaagaaacc agggactaat
gattataggc ctgtccagga tctgagagaa gtcaacaagc 3540gggtggaaga
catccacccc accgtgccca acccttacaa cctcttgagc gggctcccac
3600cgtcccacca gtggtacact gtgcttgatt taaaggatgc ctttttctgc
ctgagactcc 3660accccaccag tcagcctctc ttcgcctttg agtggagaga
tccagagatg ggaatctcag 3720gacaattgac ctggaccaga ctcccacagg
gtttcaaaaa cagtcccacc ctgtttgatg 3780aggcactgca cagagaccta
gcagacttcc ggatccagca cccagacttg atcctgctac 3840agtacgtgga
tgacttactg ctggccgcca cttctgagct agactgccaa caaggtactc
3900gggccctgtt acaaacccta gggaacctcg ggtatcgggc ctcggccaag
aaagcccaaa 3960tttgccagaa acaggtcaag tatctggggt atcttctaaa
agagggtcag agatggctga 4020ctgaggccag aaaagagact gtgatggggc
agcctactcc gaagacccct cgacaactaa 4080gggagttcct agggacggca
ggcttctgtc gcctctggat ccctgggttt gcagaaatgg 4140cagccccctt
gtaccctctc accaaaacgg ggactctgtt taattggggc ccagaccaac
4200aaaaggccta tcaagaaatc aagcaagctc ttctaactgc cccagccctg
gggttgccag 4260atttgactaa gccctttgaa ctctttgtcg acgagaagca
gggctacgcc aaaggtgtcc 4320taacgcaaaa actgggacct tggcgtcggc
cggtggccta cctgtccaaa aagctagacc 4380cagtagcagc tgggtggccc
ccttgcctac ggatggtagc agccattgcc gtactgacaa 4440aggatgcagg
caagctaacc atgggacagc cactagtcat tctggccccc catgcagtag
4500aggcactagt caaacaaccc cccgaccgct ggctttccaa cgcccggatg
actcactatc 4560aggccttgct tttggacacg gaccgggtcc agttcggacc
ggtggtagcc ctgaacccgg 4620ctacgctgct cccactgcct gaggaagggc
tgcaacacaa ctgccttgat atcctggccg 4680aagcccacgg aacccgaccc
gacctaacgg accagccgct cccagacgcc gaccacacct 4740ggtacacgga
tggaagcagt ctcttacaag agggacagcg taaggcggga gctgcggtga
4800ccaccgagac cgaggtaatc tgggctaaag ccctgccagc cgggacatcc
gctcagcggg 4860ctgaactgat agcactcacc caggccctaa agatggcaga
aggtaagaag ctaaatgttt 4920atactgatag ccgttatgct tttgctactg
cccatatcca tggagaaata tacagaaggc 4980gtgggttgct cacatcagaa
ggcaaagaga tcaaaaataa agacgagatc ttggccctac 5040taaaagccct
ctttctgccc aaaagactta gcataatcca ttgtccagga catcaaaagg
5100gacacagcgc cgaggctaga ggcaaccgga tggctgacca agcggcccga
aaggcagcca 5160tcacagagac tccagacacc tctaccctcc tcatagaaaa
ttcatcaccc tacacctcag 5220aacattttca ttacacagtg actgatataa
aggacctaac caagttgggg gccatttatg 5280ataaaacaaa gaagtattgg
gtctaccaag gaaaacctgt gatgcctgac cagtttactt 5340ttgaattatt
agactttctt catcagctga ctcacctcag cttctcaaaa atgaaggctc
5400tcctagagag aagccacagt ccctactaca tgctgaaccg ggatcgaaca
ctcaaaaata 5460tcactgagac ctgcaaagct tgtgcacaag tcaacgccag
caagtctgcc gttaaacagg 5520gaactagggt ccgcgggcat cggcccggca
ctcattggga gatcgatttc accgagataa 5580agcccggatt gtatggctat
aaatatcttc tagtttttat agataccttt tctggctgga 5640tagaagcctt
cccaaccaag aaagaaaccg ccaaggtcgt aaccaagaag ctactagagg
5700agatcttccc caggttcggc atgcctcagg tattgggaac tgacaatggg
cctgccttcg 5760tctccaaggt gagtcagaca gtggccgatc tgttggggat
tgattggaaa ttacattgtg 5820catacagacc ccaaagctca ggccaggtag
aaagaatgaa tagaaccatc aaggagactt 5880taactaaatt aacgcttgca
actggctcta gagactgggt gctcctactc cccttagccc 5940tgtaccgagc
ccgcaacacg ccgggccccc atggcctcac cccatatgag atcttatatg
6000gggcaccccc gccccttgta aacttccctg accctgacat gacaagagtt
actaacagcc 6060cctctctcca agctcactta caggctctct acttagtcca
gcacgaagtc tggagacctc 6120tggcggcagc ctaccaagaa caactggacc
gaccggtggt acctcaccct taccgagtcg 6180gcgacacagt gtgggtccgc
cgacaccaga ctaagaacct agaacctcgc tggaaaggac 6240cttacacagt
cctgctgacc acccccaccg ccctcaaagt agacggcatc gcagcttgga
6300tacacgccgc ccacgtgaag gctgccgacc ccgggggtgg accatcctct
agactgacat 6360ggcgcgttca acgctctcaa aaccccctca agataagatt
aacccgtgga agcccttaat 6420agtcatggga gtcctgttag gagtagggat
ggcagagagc ccccatcagg tctttaatgt 6480aacctggaga gtcaccaacc
tgatgactgg gcgtaccgcc aatgccacct ccctcctggg 6540aactgtacaa
gatgccttcc caaaattata ttttgatcta tgtgatctgg tcggagagga
6600gtgggaccct tcagaccagg aaccgtatgt cgggtatggc tgcaagtacc
ccgcagggag 6660acagcggacc cggacttttg acttttacgt gtgccctggg
cataccgtaa agtcggggtg 6720tgggggacca ggagagggct actgtggtaa
atgggggtgt gaaaccaccg gacaggctta 6780ctggaagccc acatcatcgt
gggacctaat ctcccttaag cgcggtaaca ccccctggga 6840cacgggatgc
tctaaagttg cctgtggccc ctgctacgac ctctccaaag tatccaattc
6900cttccaaggg gctactcgag ggggcagatg caaccctcta gtcctagaat
tcactgatgc 6960aggaaaaaag gctaactggg acgggcccaa atcgtgggga
ctgagactgt accggacagg 7020aacagatcct attaccatgt tctccctgac
ccggcaggtc cttaatgtgg gaccccgagt 7080ccccataggg cccaacccag
tattacccga ccaaagactc ccttcctcac caatagagat 7140tgtaccggct
ccacagccac ctagccccct caataccagt tacccccctt ccactaccag
7200tacaccctca acctccccta caagtccaag tgtcccacag ccacccccag
gaactggaga 7260tagactacta gctctagtca aaggagccta tcaggcgctt
aacctcacca atcccgacaa 7320gacccaagaa tgttggctgt gcttagtgtc
gggacctcct tattacgaag gagtagcggt 7380cgtgggcact tataccaatc
attccaccgc tccggccaac tgtacggcca cttcccaaca 7440taagcttacc
ctatctgaag tgacaggaca gggcctatgc atgggggcag tacctaaaac
7500tcaccaggcc ttatgtaaca ccacccaaag cgccggctca ggatcctact
accttgcagc 7560acccgccgga acaatgtggg cttgcagcac tggattgact
ccctgcttgt ccaccacggt 7620gctcaatcta accacagatt attgtgtatt
agttgaactc tggcccagag taatttacca 7680ctcccccgat tatatgtatg
gtcagcttga acagcgtacc aaatataaaa gagagccagt 7740atcattgacc
ctggcccttc tactaggagg attaaccatg ggagggattg cagctggaat
7800agggacgggg accactgcct taattaaaac ccagcagttt gagcagcttc
atgccgctat 7860ccagacagac ctcaacgaag tcgaaaagtc aattaccaac
ctagaaaagt cactgacctc 7920gttgtctgaa gtagtcctac agaaccgcag
aggcctagat ttgctattcc taaaggaggg 7980aggtctctgc gcagccctaa
aagaagaatg ttgtttttat gcagaccaca cggggctagt 8040gagagacagc
atggccaaat taagagaaag gcttaatcag agacaaaaac tatttgagac
8100aggccaagga tggttcgaag ggctgtttaa tagatccccc tggtttacca
ccttaatctc 8160caccatcatg ggacctctaa tagtactctt actgatctta
ctctttggac cttgcattct 8220caatcgattg gtccaatttg ttaaagacag
gatctcagtg gtccaggctc tggttttgac 8280tcagcaatat caccagctaa
aacccataga gtacgagcca tgaacgcgtt actggccgaa
8340gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt attttccacc
atattgccgt 8400cttttggcaa tgtgagggcc cggaaacctg gccctgtctt
cttgacgagc attcctaggg 8460gtctttcccc tctcgccaaa ggaatgcaag
gtctgttgaa tgtcgtgaag gaagcagttc 8520ctctggaagc ttcttgaaga
caaacaacgt ctgtagcgac cctttgcagg cagcggaacc 8580ccccacctgg
cgacaggtgc ctctgcggcc aaaagccacg tgtataagat acacctgcaa
8640aggcggcaca accccagtgc cacgttgtga gttggatagt tgtggaaaga
gtcaaatggc 8700tctcctcaag cgtattcaac aaggggctga aggatgccca
gaaggtaccc cattgtatgg 8760gatctgatct ggggcctcgg tgcacatgct
ttacatgtgt ttagtcgagg ttaaaaaaac 8820gtctaggccc cccgaaccac
ggggacgtgg ttttcctttg aaaaacacga ttataaatgg 8880tgaccggcgg
catggcctcc aagtgggatc aaaagggcat ggatatcgct tacgaggagg
8940ccctgctggg ctacaaggag ggcggcgtgc ctatcggcgg ctgtctgatc
aacaacaagg 9000acggcagtgt gctgggcagg ggccacaaca tgaggttcca
gaagggctcc gccaccctgc 9060acggcgagat ctccaccctg gagaactgtg
gcaggctgga gggcaaggtg tacaaggaca 9120ccaccctgta caccaccctg
tccccttgtg acatgtgtac cggcgctatc atcatgtacg 9180gcatccctag
gtgtgtgatc ggcgagaacg tgaacttcaa gtccaagggc gagaagtacc
9240tgcaaaccag gggccacgag gtggtggttg ttgacgatga gaggtgtaag
aagctgatga 9300agcagttcat cgacgagagg cctcaggact ggttcgagga
tatcggcgag taagcggccg 9360cagataaaat aaaagatttt atttagtctc
cagaaaaagg ggggaatgaa agaccccacc 9420tgtaggtttg gcaagctagc
ttaagtaacg ccattttgca aggcatggaa aaatacataa 9480ctgagaatag
agaagttcag atcaaggtca ggaacagatg gaacagctga atatgggcca
9540aacaggatat ctgtggtaag cagttcctgc cccggctcag ggccaagaac
agatggaaca 9600gctgaatatg ggccaaacag gatatctgtg gtaagcagtt
cctgccccgg ctcagggcca 9660agaacagatg gtccccagat gcggtccagc
cctcagcagt ttctagagaa ccatcagatg 9720tttccagggt gccccaagga
cctgaaatga ccctgtgcct tatttgaact aaccaatcag 9780ttcgcttctc
gcttctgttc gcgcgcttct gctccccgag ctcaataaaa gagcccacaa
9840cccctcactc ggggcgccag tcctccgatt gactgagtcg cccgggtacc
cgtgtatcca 9900ataaaccctc ttgcagttgc atccgacttg tggtctcgct
gttccttggg agggtctcct 9960ctgagtgatt gactacccgt cagcgggggt
ctttcattac atgtgagcaa aaggccagca 10020aaaggccagg aaccgtaaaa
aggccgcgtt gctggcgttt ttccataggc tccgcccccc 10080tgacgagcat
cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
10140aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
cgaccctgcc 10200gcttaccgga tacctgtccg cctttctccc ttcgggaagc
gtggcgcttt ctcaatgctc 10260acgctgtagg tatctcagtt cggtgtaggt
cgttcgctcc aagctgggct gtgtgcacga 10320accccccgtt cagcccgacc
gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 10380ggtaagacac
gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
10440gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
acactagaag 10500gacagtattt ggtatctgcg ctctgctgaa gccagttacc
ttcggaaaaa gagttggtag 10560ctcttgatcc ggcaaacaaa ccaccgctgg
tagcggtggt ttttttgttt gcaagcagca 10620gattacgcgc agaaaaaaag
gatctcaaga agatcctttg atcttttcta cggggtctga 10680cgctcagtgg
aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
10740cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
gtatatatga 10800gtaaacttgg tctgacagtt accaatgctt aatcagtgag
gcacctatct cagcgatctg 10860tctatttcgt tcatccatag ttgcctgact
ccccgtcgtg tagataacta cgatacggga 10920gggcttacca tctggcccca
gtgctgcaat gataccgcga gacccacgct caccggctcc 10980agatttatca
gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
11040tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
gtagttcgcc 11100agttaatagt ttgcgcaacg ttgttgccat tgctgcaggc
atcgtggtgt cacgctcgtc 11160gtttggtatg gcttcattca gctccggttc
ccaacgatca aggcgagtta catgatcccc 11220catgttgtgc aaaaaagcgg
ttagctcctt cggtcctccg atcgttgtca gaagtaagtt 11280ggccgcagtg
ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
11340atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
gagaatagtg 11400tatgcggcga ccgagttgct cttgcccggc gtcaacacgg
gataataccg cgccacatag 11460cagaacttta aaagtgctca tcattggaaa
acgttcttcg gggcgaaaac tctcaaggat 11520cttaccgctg ttgagatcca
gttcgatgta acccactcgt gcacccaact gatcttcagc 11580atcttttact
ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
11640aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
ttcaatatta 11700ttgaagcatt tatcagggtt attgtctcat gagcggatac
atatttgaat gtatttagaa 11760aaataaacaa ataggggttc cgcgcacatt
tccccgaaaa gtgccacctg acgtctaaga 11820aaccattatt atcatgacat
taacctataa aaataggcgt atcacgaggc cctttcgtct 11880tcaagaattc at
118922011892DNAArtificial SequenceRCR Vector - pAC3-yCD
20tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
60cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
120gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
attgacgtca 180atgggtggag tatttacggt aaactgccca cttggcagta
catcaagtgt atcatatgcc 240aagtacgccc cctattgacg tcaatgacgg
taaatggccc gcctggcatt atgcccagta 300catgacctta tgggactttc
ctacttggca gtacatctac gtattagtca tcgctattac 360catggtgatg
cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
420atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
aaaatcaacg 480ggactttcca aaatgtcgta acaactccgc cccattgacg
caaatgggcg gtaggcgtgt 540acggtgggag gtctatataa gcagagctgg
tttagtgaac cggcgccagt cctccgattg 600actgagtcgc ccgggtaccc
gtgtatccaa taaaccctct tgcagttgca tccgacttgt 660ggtctcgctg
ttccttggga gggtctcctc tgagtgattg actacccgtc agcgggggtc
720tttcatttgg gggctcgtcc gggatcggga gacccctgcc cagggaccac
cgacccacca 780ccgggaggta agctggccag caacttatct gtgtctgtcc
gattgtctag tgtctatgac 840tgattttatg cgcctgcgtc ggtactagtt
agctaactag ctctgtatct ggcggacccg 900tggtggaact gacgagttcg
gaacacccgg ccgcaaccct gggagacgtc ccagggactt 960cgggggccgt
ttttgtggcc cgacctgagt ccaaaaatcc cgatcgtttt ggactctttg
1020gtgcaccccc cttagaggag ggatatgtgg ttctggtagg agacgagaac
ctaaaacagt 1080tcccgcctcc gtctgaattt ttgctttcgg tttgggaccg
aagccgcgcc gcgcgtcttg 1140tctgctgcag catcgttctg tgttgtctct
gtctgactgt gtttctgtat ttgtctgaga 1200atatgggcca gactgttacc
actcccttaa gtttgacctt aggtcactgg aaagatgtcg 1260agcggatcgc
tcacaaccag tcggtagatg tcaagaagag acgttgggtt accttctgct
1320ctgcagaatg gccaaccttt aacgtcggat ggccgcgaga cggcaccttt
aaccgagacc 1380tcatcaccca ggttaagatc aaggtctttt cacctggccc
gcatggacac ccagaccagg 1440tcccctacat cgtgacctgg gaagccttgg
cttttgaccc ccctccctgg gtcaagccct 1500ttgtacaccc taagcctccg
cctcctcttc ctccatccgc cccgtctctc ccccttgaac 1560ctcctcgttc
gaccccgcct cgatcctccc tttatccagc cctcactcct tctctaggcg
1620ccaaacctaa acctcaagtt ctttctgaca gtggggggcc gctcatcgac
ctacttacag 1680aagacccccc gccttatagg gacccaagac cacccccttc
cgacagggac ggaaatggtg 1740gagaagcgac ccctgcggga gaggcaccgg
acccctcccc aatggcatct cgcctacgtg 1800ggagacggga gccccctgtg
gccgactcca ctacctcgca ggcattcccc ctccgcgcag 1860gaggaaacgg
acagcttcaa tactggccgt tctcctcttc tgacctttac aactggaaaa
1920ataataaccc ttctttttct gaagatccag gtaaactgac agctctgatc
gagtctgttc 1980tcatcaccca tcagcccacc tgggacgact gtcagcagct
gttggggact ctgctgaccg 2040gagaagaaaa acaacgggtg ctcttagagg
ctagaaaggc ggtgcggggc gatgatgggc 2100gccccactca actgcccaat
gaagtcgatg ccgcttttcc cctcgagcgc ccagactggg 2160attacaccac
ccaggcaggt aggaaccacc tagtccacta tcgccagttg ctcctagcgg
2220gtctccaaaa cgcgggcaga agccccacca atttggccaa ggtaaaagga
ataacacaag 2280ggcccaatga gtctccctcg gccttcctag agagacttaa
ggaagcctat cgcaggtaca 2340ctccttatga ccctgaggac ccagggcaag
aaactaatgt gtctatgtct ttcatttggc 2400agtctgcccc agacattggg
agaaagttag agaggttaga agatttaaaa aacaagacgc 2460ttggagattt
ggttagagag gcagaaaaga tctttaataa acgagaaacc ccggaagaaa
2520gagaggaacg tatcaggaga gaaacagagg aaaaagaaga acgccgtagg
acagaggatg 2580agcagaaaga gaaagaaaga gatcgtagga gacatagaga
gatgagcaag ctattggcca 2640ctgtcgttag tggacagaaa caggatagac
agggaggaga acgaaggagg tcccaactcg 2700atcgcgacca gtgtgcctac
tgcaaagaaa aggggcactg ggctaaagat tgtcccaaga 2760aaccacgagg
acctcgggga ccaagacccc agacctccct cctgacccta gatgactagg
2820gaggtcaggg tcaggagccc ccccctgaac ccaggataac cctcaaagtc
ggggggcaac 2880ccgtcacctt cctggtagat actggggccc aacactccgt
gctgacccaa aatcctggac 2940ccctaagtga taagtctgcc tgggtccaag
gggctactgg aggaaagcgg tatcgctgga 3000ccacggatcg caaagtacat
ctagctaccg gtaaggtcac ccactctttc ctccatgtac 3060cagactgtcc
ctatcctctg ttaggaagag atttgctgac taaactaaaa gcccaaatcc
3120actttgaggg atcaggagcc caggttatgg gaccaatggg gcagcccctg
caagtgttga 3180ccctaaatat agaagatgag catcggctac atgagacctc
aaaagagcca gatgtttctc 3240tagggtccac atggctgtct gattttcctc
aggcctgggc ggaaaccggg ggcatgggac 3300tggcagttcg ccaagctcct
ctgatcatac ctctgaaagc aacctctacc cccgtgtcca 3360taaaacaata
ccccatgtca caagaagcca gactggggat caagccccac atacagagac
3420tgttggacca gggaatactg gtaccctgcc agtccccctg gaacacgccc
ctgctacccg 3480ttaagaaacc agggactaat gattataggc ctgtccagga
tctgagagaa gtcaacaagc 3540gggtggaaga catccacccc accgtgccca
acccttacaa cctcttgagc gggctcccac 3600cgtcccacca gtggtacact
gtgcttgatt taaaggatgc ctttttctgc ctgagactcc 3660accccaccag
tcagcctctc ttcgcctttg agtggagaga tccagagatg ggaatctcag
3720gacaattgac ctggaccaga ctcccacagg gtttcaaaaa cagtcccacc
ctgtttgatg 3780aggcactgca cagagaccta gcagacttcc ggatccagca
cccagacttg atcctgctac 3840agtacgtgga tgacttactg ctggccgcca
cttctgagct agactgccaa caaggtactc 3900gggccctgtt acaaacccta
gggaacctcg ggtatcgggc ctcggccaag aaagcccaaa 3960tttgccagaa
acaggtcaag tatctggggt atcttctaaa agagggtcag agatggctga
4020ctgaggccag aaaagagact gtgatggggc agcctactcc gaagacccct
cgacaactaa 4080gggagttcct agggacggca ggcttctgtc gcctctggat
ccctgggttt gcagaaatgg 4140cagccccctt gtaccctctc accaaaacgg
ggactctgtt taattggggc ccagaccaac 4200aaaaggccta tcaagaaatc
aagcaagctc ttctaactgc cccagccctg gggttgccag 4260atttgactaa
gccctttgaa ctctttgtcg acgagaagca gggctacgcc aaaggtgtcc
4320taacgcaaaa actgggacct tggcgtcggc cggtggccta cctgtccaaa
aagctagacc 4380cagtagcagc tgggtggccc ccttgcctac ggatggtagc
agccattgcc gtactgacaa 4440aggatgcagg caagctaacc atgggacagc
cactagtcat tctggccccc catgcagtag 4500aggcactagt caaacaaccc
cccgaccgct ggctttccaa cgcccggatg actcactatc 4560aggccttgct
tttggacacg gaccgggtcc agttcggacc ggtggtagcc ctgaacccgg
4620ctacgctgct cccactgcct gaggaagggc tgcaacacaa ctgccttgat
atcctggccg 4680aagcccacgg aacccgaccc gacctaacgg accagccgct
cccagacgcc gaccacacct 4740ggtacacgga tggaagcagt ctcttacaag
agggacagcg taaggcggga gctgcggtga 4800ccaccgagac cgaggtaatc
tgggctaaag ccctgccagc cgggacatcc gctcagcggg 4860ctgaactgat
agcactcacc caggccctaa agatggcaga aggtaagaag ctaaatgttt
4920atactgatag ccgttatgct tttgctactg cccatatcca tggagaaata
tacagaaggc 4980gtgggttgct cacatcagaa ggcaaagaga tcaaaaataa
agacgagatc ttggccctac 5040taaaagccct ctttctgccc aaaagactta
gcataatcca ttgtccagga catcaaaagg 5100gacacagcgc cgaggctaga
ggcaaccgga tggctgacca agcggcccga aaggcagcca 5160tcacagagac
tccagacacc tctaccctcc tcatagaaaa ttcatcaccc tacacctcag
5220aacattttca ttacacagtg actgatataa aggacctaac caagttgggg
gccatttatg 5280ataaaacaaa gaagtattgg gtctaccaag gaaaacctgt
gatgcctgac cagtttactt 5340ttgaattatt agactttctt catcagctga
ctcacctcag cttctcaaaa atgaaggctc 5400tcctagagag aagccacagt
ccctactaca tgctgaaccg ggatcgaaca ctcaaaaata 5460tcactgagac
ctgcaaagct tgtgcacaag tcaacgccag caagtctgcc gttaaacagg
5520gaactagggt ccgcgggcat cggcccggca ctcattggga gatcgatttc
accgagataa 5580agcccggatt gtatggctat aaatatcttc tagtttttat
agataccttt tctggctgga 5640tagaagcctt cccaaccaag aaagaaaccg
ccaaggtcgt aaccaagaag ctactagagg 5700agatcttccc caggttcggc
atgcctcagg tattgggaac tgacaatggg cctgccttcg 5760tctccaaggt
gagtcagaca gtggccgatc tgttggggat tgattggaaa ttacattgtg
5820catacagacc ccaaagctca ggccaggtag aaagaatgaa tagaaccatc
aaggagactt 5880taactaaatt aacgcttgca actggctcta gagactgggt
gctcctactc cccttagccc 5940tgtaccgagc ccgcaacacg ccgggccccc
atggcctcac cccatatgag atcttatatg 6000gggcaccccc gccccttgta
aacttccctg accctgacat gacaagagtt actaacagcc 6060cctctctcca
agctcactta caggctctct acttagtcca gcacgaagtc tggagacctc
6120tggcggcagc ctaccaagaa caactggacc gaccggtggt acctcaccct
taccgagtcg 6180gcgacacagt gtgggtccgc cgacaccaga ctaagaacct
agaacctcgc tggaaaggac 6240cttacacagt cctgctgacc acccccaccg
ccctcaaagt agacggcatc gcagcttgga 6300tacacgccgc ccacgtgaag
gctgccgacc ccgggggtgg accatcctct agactgacat 6360ggcgcgttca
acgctctcaa aaccccctca agataagatt aacccgtgga agcccttaat
6420agtcatggga gtcctgttag gagtagggat ggcagagagc ccccatcagg
tctttaatgt 6480aacctggaga gtcaccaacc tgatgactgg gcgtaccgcc
aatgccacct ccctcctggg 6540aactgtacaa gatgccttcc caaaattata
ttttgatcta tgtgatctgg tcggagagga 6600gtgggaccct tcagaccagg
aaccgtatgt cgggtatggc tgcaagtacc ccgcagggag 6660acagcggacc
cggacttttg acttttacgt gtgccctggg cataccgtaa agtcggggtg
6720tgggggacca ggagagggct actgtggtaa atgggggtgt gaaaccaccg
gacaggctta 6780ctggaagccc acatcatcgt gggacctaat ctcccttaag
cgcggtaaca ccccctggga 6840cacgggatgc tctaaagttg cctgtggccc
ctgctacgac ctctccaaag tatccaattc 6900cttccaaggg gctactcgag
ggggcagatg caaccctcta gtcctagaat tcactgatgc 6960aggaaaaaag
gctaactggg acgggcccaa atcgtgggga ctgagactgt accggacagg
7020aacagatcct attaccatgt tctccctgac ccggcaggtc cttaatgtgg
gaccccgagt 7080ccccataggg cccaacccag tattacccga ccaaagactc
ccttcctcac caatagagat 7140tgtaccggct ccacagccac ctagccccct
caataccagt tacccccctt ccactaccag 7200tacaccctca acctccccta
caagtccaag tgtcccacag ccacccccag gaactggaga 7260tagactacta
gctctagtca aaggagccta tcaggcgctt aacctcacca atcccgacaa
7320gacccaagaa tgttggctgt gcttagtgtc gggacctcct tattacgaag
gagtagcggt 7380cgtgggcact tataccaatc attccaccgc tccggccaac
tgtacggcca cttcccaaca 7440taagcttacc ctatctgaag tgacaggaca
gggcctatgc atgggggcag tacctaaaac 7500tcaccaggcc ttatgtaaca
ccacccaaag cgccggctca ggatcctact accttgcagc 7560acccgccgga
acaatgtggg cttgcagcac tggattgact ccctgcttgt ccaccacggt
7620gctcaatcta accacagatt attgtgtatt agttgaactc tggcccagag
taatttacca 7680ctcccccgat tatatgtatg gtcagcttga acagcgtacc
aaatataaaa gagagccagt 7740atcattgacc ctggcccttc tactaggagg
attaaccatg ggagggattg cagctggaat 7800agggacgggg accactgcct
taattaaaac ccagcagttt gagcagcttc atgccgctat 7860ccagacagac
ctcaacgaag tcgaaaagtc aattaccaac ctagaaaagt cactgacctc
7920gttgtctgaa gtagtcctac agaaccgcag aggcctagat ttgctattcc
taaaggaggg 7980aggtctctgc gcagccctaa aagaagaatg ttgtttttat
gcagaccaca cggggctagt 8040gagagacagc atggccaaat taagagaaag
gcttaatcag agacaaaaac tatttgagac 8100aggccaagga tggttcgaag
ggctgtttaa tagatccccc tggtttacca ccttaatctc 8160caccatcatg
ggacctctaa tagtactctt actgatctta ctctttggac cttgcattct
8220caatcgattg gtccaatttg ttaaagacag gatctcagtg gtccaggctc
tggttttgac 8280tcagcaatat caccagctaa aacccataga gtacgagcca
tgaacgcgtt actggccgaa 8340gccgcttgga ataaggccgg tgtgcgtttg
tctatatgtt attttccacc atattgccgt 8400cttttggcaa tgtgagggcc
cggaaacctg gccctgtctt cttgacgagc attcctaggg 8460gtctttcccc
tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc
8520ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg
cagcggaacc 8580ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg
tgtataagat acacctgcaa 8640aggcggcaca accccagtgc cacgttgtga
gttggatagt tgtggaaaga gtcaaatggc 8700tctcctcaag cgtattcaac
aaggggctga aggatgccca gaaggtaccc cattgtatgg 8760gatctgatct
ggggcctcgg tgcacatgct ttacatgtgt ttagtcgagg ttaaaaaaac
8820gtctaggccc cccgaaccac ggggacgtgg ttttcctttg aaaaacacga
ttataaatgg 8880tgacaggggg aatggcaagc aagtgggatc agaagggtat
ggacattgcc tatgaggagg 8940cggccttagg ttacaaagag ggtggtgttc
ctattggcgg atgtcttatc aataacaaag 9000acggaagtgt tctcggtcgt
ggtcacaaca tgagatttca aaagggatcc gccacactac 9060atggtgagat
ctccactttg gaaaactgtg ggagattaga gggcaaagtg tacaaagata
9120ccactttgta tacgacgctg tctccatgcg acatgtgtac aggtgccatc
atcatgtatg 9180gtattccacg ctgtgttgtc ggtgagaacg ttaatttcaa
aagtaagggc gagaaatatt 9240tacaaactag aggtcacgag gttgttgttg
ttgacgatga gaggtgtaaa aagatcatga 9300aacaatttat cgatgaaaga
cctcaggatt ggtttgaaga tattggtgag taggcggccg 9360cagataaaat
aaaagatttt atttagtctc cagaaaaagg ggggaatgaa agaccccacc
9420tgtaggtttg gcaagctagc ttaagtaacg ccattttgca aggcatggaa
aaatacataa 9480ctgagaatag agaagttcag atcaaggtca ggaacagatg
gaacagctga atatgggcca 9540aacaggatat ctgtggtaag cagttcctgc
cccggctcag ggccaagaac agatggaaca 9600gctgaatatg ggccaaacag
gatatctgtg gtaagcagtt cctgccccgg ctcagggcca 9660agaacagatg
gtccccagat gcggtccagc cctcagcagt ttctagagaa ccatcagatg
9720tttccagggt gccccaagga cctgaaatga ccctgtgcct tatttgaact
aaccaatcag 9780ttcgcttctc gcttctgttc gcgcgcttct gctccccgag
ctcaataaaa gagcccacaa 9840cccctcactc ggggcgccag tcctccgatt
gactgagtcg cccgggtacc cgtgtatcca 9900ataaaccctc ttgcagttgc
atccgacttg tggtctcgct gttccttggg agggtctcct 9960ctgagtgatt
gactacccgt cagcgggggt ctttcattac atgtgagcaa aaggccagca
10020aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
tccgcccccc 10080tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
cgaaacccga caggactata 10140aagataccag gcgtttcccc ctggaagctc
cctcgtgcgc tctcctgttc cgaccctgcc 10200gcttaccgga tacctgtccg
cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc 10260acgctgtagg
tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
10320accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
agtccaaccc 10380ggtaagacac gacttatcgc cactggcagc agccactggt
aacaggatta gcagagcgag 10440gtatgtaggc ggtgctacag agttcttgaa
gtggtggcct aactacggct acactagaag 10500gacagtattt ggtatctgcg
ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 10560ctcttgatcc
ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
10620gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
cggggtctga 10680cgctcagtgg aacgaaaact cacgttaagg gattttggtc
atgagattat caaaaaggat 10740cttcacctag atccttttaa attaaaaatg
aagttttaaa tcaatctaaa gtatatatga 10800gtaaacttgg tctgacagtt
accaatgctt aatcagtgag gcacctatct cagcgatctg 10860tctatttcgt
tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
10920gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
caccggctcc 10980agatttatca gcaataaacc agccagccgg aagggccgag
cgcagaagtg gtcctgcaac 11040tttatccgcc tccatccagt ctattaattg
ttgccgggaa gctagagtaa gtagttcgcc 11100agttaatagt ttgcgcaacg
ttgttgccat tgctgcaggc atcgtggtgt cacgctcgtc 11160gtttggtatg
gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
11220catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
gaagtaagtt 11280ggccgcagtg ttatcactca tggttatggc agcactgcat
aattctctta ctgtcatgcc 11340atccgtaaga tgcttttctg tgactggtga
gtactcaacc aagtcattct gagaatagtg 11400tatgcggcga
ccgagttgct cttgcccggc gtcaacacgg gataataccg cgccacatag
11460cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
tctcaaggat 11520cttaccgctg ttgagatcca gttcgatgta acccactcgt
gcacccaact gatcttcagc 11580atcttttact ttcaccagcg tttctgggtg
agcaaaaaca ggaaggcaaa atgccgcaaa 11640aaagggaata agggcgacac
ggaaatgttg aatactcata ctcttccttt ttcaatatta 11700ttgaagcatt
tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
11760aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
acgtctaaga 11820aaccattatt atcatgacat taacctataa aaataggcgt
atcacgaggc cctttcgtct 11880tcaagaattc at 118922112007DNAArtificial
SequenceRCR Vector - pACE-CD 21tagttattaa tagtaatcaa ttacggggtc
attagttcat agcccatata tggagttccg 60cgttacataa cttacggtaa atggcccgcc
tggctgaccg cccaacgacc cccgcccatt 120gacgtcaata atgacgtatg
ttcccatagt aacgccaata gggactttcc attgacgtca 180atgggtggag
tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
240aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
atgcccagta 300catgacctta tgggactttc ctacttggca gtacatctac
gtattagtca tcgctattac 360catggtgatg cggttttggc agtacatcaa
tgggcgtgga tagcggtttg actcacgggg 420atttccaagt ctccacccca
ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 480ggactttcca
aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
540acggtgggag gtctatataa gcagagctgg tttagtgaac cggcgccagt
cctccgattg 600actgagtcgc ccgggtaccc gtgtatccaa taaaccctct
tgcagttgca tccgacttgt 660ggtctcgctg ttccttggga gggtctcctc
tgagtgattg actacccgtc agcgggggtc 720tttcatttgg gggctcgtcc
gggatcggga gacccctgcc cagggaccac cgacccacca 780ccgggaggta
agctggccag caacttatct gtgtctgtcc gattgtctag tgtctatgac
840tgattttatg cgcctgcgtc ggtactagtt agctaactag ctctgtatct
ggcggacccg 900tggtggaact gacgagttcg gaacacccgg ccgcaaccct
gggagacgtc ccagggactt 960cgggggccgt ttttgtggcc cgacctgagt
ccaaaaatcc cgatcgtttt ggactctttg 1020gtgcaccccc cttagaggag
ggatatgtgg ttctggtagg agacgagaac ctaaaacagt 1080tcccgcctcc
gtctgaattt ttgctttcgg tttgggaccg aagccgcgcc gcgcgtcttg
1140tctgctgcag catcgttctg tgttgtctct gtctgactgt gtttctgtat
ttgtctgaga 1200atatgggcca gactgttacc actcccttaa gtttgacctt
aggtcactgg aaagatgtcg 1260agcggatcgc tcacaaccag tcggtagatg
tcaagaagag acgttgggtt accttctgct 1320ctgcagaatg gccaaccttt
aacgtcggat ggccgcgaga cggcaccttt aaccgagacc 1380tcatcaccca
ggttaagatc aaggtctttt cacctggccc gcatggacac ccagaccagg
1440tcccctacat cgtgacctgg gaagccttgg cttttgaccc ccctccctgg
gtcaagccct 1500ttgtacaccc taagcctccg cctcctcttc ctccatccgc
cccgtctctc ccccttgaac 1560ctcctcgttc gaccccgcct cgatcctccc
tttatccagc cctcactcct tctctaggcg 1620ccaaacctaa acctcaagtt
ctttctgaca gtggggggcc gctcatcgac ctacttacag 1680aagacccccc
gccttatagg gacccaagac cacccccttc cgacagggac ggaaatggtg
1740gagaagcgac ccctgcggga gaggcaccgg acccctcccc aatggcatct
cgcctacgtg 1800ggagacggga gccccctgtg gccgactcca ctacctcgca
ggcattcccc ctccgcgcag 1860gaggaaacgg acagcttcaa tactggccgt
tctcctcttc tgacctttac aactggaaaa 1920ataataaccc ttctttttct
gaagatccag gtaaactgac agctctgatc gagtctgttc 1980tcatcaccca
tcagcccacc tgggacgact gtcagcagct gttggggact ctgctgaccg
2040gagaagaaaa acaacgggtg ctcttagagg ctagaaaggc ggtgcggggc
gatgatgggc 2100gccccactca actgcccaat gaagtcgatg ccgcttttcc
cctcgagcgc ccagactggg 2160attacaccac ccaggcaggt aggaaccacc
tagtccacta tcgccagttg ctcctagcgg 2220gtctccaaaa cgcgggcaga
agccccacca atttggccaa ggtaaaagga ataacacaag 2280ggcccaatga
gtctccctcg gccttcctag agagacttaa ggaagcctat cgcaggtaca
2340ctccttatga ccctgaggac ccagggcaag aaactaatgt gtctatgtct
ttcatttggc 2400agtctgcccc agacattggg agaaagttag agaggttaga
agatttaaaa aacaagacgc 2460ttggagattt ggttagagag gcagaaaaga
tctttaataa acgagaaacc ccggaagaaa 2520gagaggaacg tatcaggaga
gaaacagagg aaaaagaaga acgccgtagg acagaggatg 2580agcagaaaga
gaaagaaaga gatcgtagga gacatagaga gatgagcaag ctattggcca
2640ctgtcgttag tggacagaaa caggatagac agggaggaga acgaaggagg
tcccaactcg 2700atcgcgacca gtgtgcctac tgcaaagaaa aggggcactg
ggctaaagat tgtcccaaga 2760aaccacgagg acctcgggga ccaagacccc
agacctccct cctgacccta gatgactagg 2820gaggtcaggg tcaggagccc
ccccctgaac ccaggataac cctcaaagtc ggggggcaac 2880ccgtcacctt
cctggtagat actggggccc aacactccgt gctgacccaa aatcctggac
2940ccctaagtga taagtctgcc tgggtccaag gggctactgg aggaaagcgg
tatcgctgga 3000ccacggatcg caaagtacat ctagctaccg gtaaggtcac
ccactctttc ctccatgtac 3060cagactgtcc ctatcctctg ttaggaagag
atttgctgac taaactaaaa gcccaaatcc 3120actttgaggg atcaggagcc
caggttatgg gaccaatggg gcagcccctg caagtgttga 3180ccctaaatat
agaagatgag catcggctac atgagacctc aaaagagcca gatgtttctc
3240tagggtccac atggctgtct gattttcctc aggcctgggc ggaaaccggg
ggcatgggac 3300tggcagttcg ccaagctcct ctgatcatac ctctgaaagc
aacctctacc cccgtgtcca 3360taaaacaata ccccatgtca caagaagcca
gactggggat caagccccac atacagagac 3420tgttggacca gggaatactg
gtaccctgcc agtccccctg gaacacgccc ctgctacccg 3480ttaagaaacc
agggactaat gattataggc ctgtccagga tctgagagaa gtcaacaagc
3540gggtggaaga catccacccc accgtgccca acccttacaa cctcttgagc
gggctcccac 3600cgtcccacca gtggtacact gtgcttgatt taaaggatgc
ctttttctgc ctgagactcc 3660accccaccag tcagcctctc ttcgcctttg
agtggagaga tccagagatg ggaatctcag 3720gacaattgac ctggaccaga
ctcccacagg gtttcaaaaa cagtcccacc ctgtttgatg 3780aggcactgca
cagagaccta gcagacttcc ggatccagca cccagacttg atcctgctac
3840agtacgtgga tgacttactg ctggccgcca cttctgagct agactgccaa
caaggtactc 3900gggccctgtt acaaacccta gggaacctcg ggtatcgggc
ctcggccaag aaagcccaaa 3960tttgccagaa acaggtcaag tatctggggt
atcttctaaa agagggtcag agatggctga 4020ctgaggccag aaaagagact
gtgatggggc agcctactcc gaagacccct cgacaactaa 4080gggagttcct
agggacggca ggcttctgtc gcctctggat ccctgggttt gcagaaatgg
4140cagccccctt gtaccctctc accaaaacgg ggactctgtt taattggggc
ccagaccaac 4200aaaaggccta tcaagaaatc aagcaagctc ttctaactgc
cccagccctg gggttgccag 4260atttgactaa gccctttgaa ctctttgtcg
acgagaagca gggctacgcc aaaggtgtcc 4320taacgcaaaa actgggacct
tggcgtcggc cggtggccta cctgtccaaa aagctagacc 4380cagtagcagc
tgggtggccc ccttgcctac ggatggtagc agccattgcc gtactgacaa
4440aggatgcagg caagctaacc atgggacagc cactagtcat tctggccccc
catgcagtag 4500aggcactagt caaacaaccc cccgaccgct ggctttccaa
cgcccggatg actcactatc 4560aggccttgct tttggacacg gaccgggtcc
agttcggacc ggtggtagcc ctgaacccgg 4620ctacgctgct cccactgcct
gaggaagggc tgcaacacaa ctgccttgat atcctggccg 4680aagcccacgg
aacccgaccc gacctaacgg accagccgct cccagacgcc gaccacacct
4740ggtacacgga tggaagcagt ctcttacaag agggacagcg taaggcggga
gctgcggtga 4800ccaccgagac cgaggtaatc tgggctaaag ccctgccagc
cgggacatcc gctcagcggg 4860ctgaactgat agcactcacc caggccctaa
agatggcaga aggtaagaag ctaaatgttt 4920atactgatag ccgttatgct
tttgctactg cccatatcca tggagaaata tacagaaggc 4980gtgggttgct
cacatcagaa ggcaaagaga tcaaaaataa agacgagatc ttggccctac
5040taaaagccct ctttctgccc aaaagactta gcataatcca ttgtccagga
catcaaaagg 5100gacacagcgc cgaggctaga ggcaaccgga tggctgacca
agcggcccga aaggcagcca 5160tcacagagac tccagacacc tctaccctcc
tcatagaaaa ttcatcaccc tacacctcag 5220aacattttca ttacacagtg
actgatataa aggacctaac caagttgggg gccatttatg 5280ataaaacaaa
gaagtattgg gtctaccaag gaaaacctgt gatgcctgac cagtttactt
5340ttgaattatt agactttctt catcagctga ctcacctcag cttctcaaaa
atgaaggctc 5400tcctagagag aagccacagt ccctactaca tgctgaaccg
ggatcgaaca ctcaaaaata 5460tcactgagac ctgcaaagct tgtgcacaag
tcaacgccag caagtctgcc gttaaacagg 5520gaactagggt ccgcgggcat
cggcccggca ctcattggga gatcgatttc accgagataa 5580agcccggatt
gtatggctat aaatatcttc tagtttttat agataccttt tctggctgga
5640tagaagcctt cccaaccaag aaagaaaccg ccaaggtcgt aaccaagaag
ctactagagg 5700agatcttccc caggttcggc atgcctcagg tattgggaac
tgacaatggg cctgccttcg 5760tctccaaggt gagtcagaca gtggccgatc
tgttggggat tgattggaaa ttacattgtg 5820catacagacc ccaaagctca
ggccaggtag aaagaatgaa tagaaccatc aaggagactt 5880taactaaatt
aacgcttgca actggctcta gagactgggt gctcctactc cccttagccc
5940tgtaccgagc ccgcaacacg ccgggccccc atggcctcac cccatatgag
atcttatatg 6000gggcaccccc gccccttgta aacttccctg accctgacat
gacaagagtt actaacagcc 6060cctctctcca agctcactta caggctctct
acttagtcca gcacgaagtc tggagacctc 6120tggcggcagc ctaccaagaa
caactggacc gaccggtggt acctcaccct taccgagtcg 6180gcgacacagt
gtgggtccgc cgacaccaga ctaagaacct agaacctcgc tggaaaggac
6240cttacacagt cctgctgacc acccccaccg ccctcaaagt agacggcatc
gcagcttgga 6300tacacgccgc ccacgtgaag gctgccgacc ccgggggtgg
accatcctct agactgacat 6360ggcgcgttca acgctctcaa aaccccctca
agataagatt aacccgtgga agcccttaat 6420agtcatggga gtcctgttag
gagtagggat ggcagagagc ccccatcagg tctttaatgt 6480aacctggaga
gtcaccaacc tgatgactgg gcgtaccgcc aatgccacct ccctcctggg
6540aactgtacaa gatgccttcc caaaattata ttttgatcta tgtgatctgg
tcggagagga 6600gtgggaccct tcagaccagg aaccgtatgt cgggtatggc
tgcaagtacc ccgcagggag 6660acagcggacc cggacttttg acttttacgt
gtgccctggg cataccgtaa agtcggggtg 6720tgggggacca ggagagggct
actgtggtaa atgggggtgt gaaaccaccg gacaggctta 6780ctggaagccc
acatcatcgt gggacctaat ctcccttaag cgcggtaaca ccccctggga
6840cacgggatgc tctaaagttg cctgtggccc ctgctacgac ctctccaaag
tatccaattc 6900cttccaaggg gctactcgag ggggcagatg caaccctcta
gtcctagaat tcactgatgc 6960aggaaaaaag gctaactggg acgggcccaa
atcgtgggga ctgagactgt accggacagg 7020aacagatcct attaccatgt
tctccctgac ccggcaggtc cttaatgtgg gaccccgagt 7080ccccataggg
cccaacccag tattacccga ccaaagactc ccttcctcac caatagagat
7140tgtaccggct ccacagccac ctagccccct caataccagt tacccccctt
ccactaccag 7200tacaccctca acctccccta caagtccaag tgtcccacag
ccacccccag gaactggaga 7260tagactacta gctctagtca aaggagccta
tcaggcgctt aacctcacca atcccgacaa 7320gacccaagaa tgttggctgt
gcttagtgtc gggacctcct tattacgaag gagtagcggt 7380cgtgggcact
tataccaatc attccaccgc tccggccaac tgtacggcca cttcccaaca
7440taagcttacc ctatctgaag tgacaggaca gggcctatgc atgggggcag
tacctaaaac 7500tcaccaggcc ttatgtaaca ccacccaaag cgccggctca
ggatcctact accttgcagc 7560acccgccgga acaatgtggg cttgcagcac
tggattgact ccctgcttgt ccaccacggt 7620gctcaatcta accacagatt
attgtgtatt agttgaactc tggcccagag taatttacca 7680ctcccccgat
tatatgtatg gtcagcttga acagcgtacc aaatataaaa gagagccagt
7740atcattgacc ctggcccttc tactaggagg attaaccatg ggagggattg
cagctggaat 7800agggacgggg accactgcct taattaaaac ccagcagttt
gagcagcttc atgccgctat 7860ccagacagac ctcaacgaag tcgaaaagtc
aattaccaac ctagaaaagt cactgacctc 7920gttgtctgaa gtagtcctac
agaaccgcag aggcctagat ttgctattcc taaaggaggg 7980aggtctctgc
gcagccctaa aagaagaatg ttgtttttat gcagaccaca cggggctagt
8040gagagacagc atggccaaat taagagaaag gcttaatcag agacaaaaac
tatttgagac 8100aggccaagga tggttcgaag ggctgtttaa tagatccccc
tggtttacca ccttaatctc 8160caccatcatg ggacctctaa tagtactctt
actgatctta ctctttggac cttgcattct 8220caatcgatta gtccaatttg
ttaaagacag gatatcagtg gtccaggctc tagttttgac 8280tcaacaatat
caccagctga agcctataga gtacgagcca tgacgtacgt tactggccga
8340agccgcttgg aataaggccg gtgtgcgttt gtctatatgt tattttccac
catattgccg 8400tcttttggca atgtgagggc ccggaaacct ggccctgtct
tcttgacgag cattcctagg 8460ggtctttccc ctctcgccaa aggaatgcaa
ggtctgttga atgtcgtgaa ggaagcagtt 8520cctctggaag cttcttgaag
acaaacaacg tctgtagcga ccctttgcag gcagcggaac 8580cccccacctg
gcgacaggtg cctctgcggc caaaagccac gtgtataaga tacacctgca
8640aaggcggcac aaccccagtg ccacgttgtg agttggatag ttgtggaaag
agtcaaatgg 8700ctctcctcaa gcgtattcaa caaggggctg aaggatgccc
agaaggtacc ccattgtatg 8760ggatctgatc tggggcctcg gtgcacatgc
tttacatgtg tttagtcgag gttaaaaaaa 8820cgtctaggcc ccccgaacca
cggggacgtg gttttccttt gaaaaacacg ataataccat 8880ggtgacaggg
ggaatggcaa gcaagtggga tcagaagggt atggacattg cctatgagga
8940ggcggcctta ggttacaaag agggtggtgt tcctattggc ggatgtctta
tcaataacaa 9000agacggaagt gttctcggtc gtggtcacaa catgagattt
caaaagggat ccgccacact 9060acatggtgag atctccactt tggaaaactg
tgggagatta gagggcaaag tgtacaaaga 9120taccactttg tatacgacgc
tgtctccatg cgacatgtgt acaggtgcca tcatcatgta 9180tggtattcca
cgctgtgttg tcggtgagaa cgttaatttc aaaagtaagg gcgagaaata
9240tttacaaact agaggtcacg aggttgttgt tgttgacgat gagaggtgta
aaaagatcat 9300gaaacaattt atcgatgaaa gacctcagga ttggtttgaa
gatattggtg agtaggcggc 9360cgcgccatag ataaaataaa agattttatt
tagtctccag aaaaaggggg gaatgaaaga 9420ccccacctgt aggtttggca
agctagctta agtaacgcca ttttgcaagg catggaaaaa 9480tacataactg
agaatagaga agttcagatc aaggtcagga acagatggaa cagctgaata
9540tgggccaaac aggatatctg tggtaagcag ttcctgcccc ggctcagggc
caagaacaga 9600tggaacagct gaatatgggc caaacaggat atctgtggta
agcagttcct gccccggctc 9660agggccaaga acagatggtc cccagatgcg
gtccagccct cagcagtttc tagagaacca 9720tcagatgttt ccagggtgcc
ccaaggacct gaaatgaccc tgtgccttgt ttaaactaac 9780caatcagttc
gcttctcgct tctgttcgcg cgcttctgct ccccgagctc aataaaagag
9840cccacaaccc ctcactcggg gcgccagtcc tccgattgac tgagtcgccc
gggtacccgt 9900gtatccaata aaccctcttg cagttgcatc cgacttgtgg
tctcgctgtt ccttgggagg 9960gtctcctctg agtgattgac tacccgtcag
cgggggtctt tcatttgggg gctcgtccgg 10020gatcgggaga cccctgccca
gggaccaccg acccaccacc gggaggtaag ctggctgcct 10080cgcgcgtttc
ggtgatgacg gtgaaaacct ctgacatgtg agcaaaaggc cagcaaaagg
10140ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
ccccctgacg 10200agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
cccgacagga ctataaagat 10260accaggcgtt tccccctgga agctccctcg
tgcgctctcc tgttccgacc ctgccgctta 10320ccggatacct gtccgccttt
ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct 10380gtaggtatct
cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
10440ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
aacccggtaa 10500gacacgactt atcgccactg gcagcagcca ctggtaacag
gattagcaga gcgaggtatg 10560taggcggtgc tacagagttc ttgaagtggt
ggcctaacta cggctacact agaaggacag 10620tatttggtat ctgcgctctg
ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt 10680gatccggcaa
acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
10740cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
tctgacgctc 10800agtggaacga aaactcacgt taagggattt tggtcatgag
attatcaaaa aggatcttca 10860cctagatcct tttaaattaa aaatgaagtt
ttaaatcaat ctaaagtata tatgagtaaa 10920cttggtctga cagttaccaa
tgcttaatca gtgaggcacc tatctcagcg atctgtctat 10980ttcgttcatc
catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
11040taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
gctccagatt 11100tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
aagtggtcct gcaactttat 11160ccgcctccat ccagtctatt aattgttgcc
gggaagctag agtaagtagt tcgccagtta 11220atagtttgcg caacgttgtt
gccattgctg caggcatcgt ggtgtcacgc tcgtcgtttg 11280gtatggcttc
attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
11340tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
aagttggccg 11400cagtgttatc actcatggtt atggcagcac tgcataattc
tcttactgtc atgccatccg 11460taagatgctt ttctgtgact ggtgagtact
caaccaagtc attctgagaa tagtgtatgc 11520ggcgaccgag ttgctcttgc
ccggcgtcaa cacgggataa taccgcgcca catagcagaa 11580ctttaaaagt
gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
11640cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
tcagcatctt 11700ttactttcac cagcgtttct gggtgagcaa aaacaggaag
gcaaaatgcc gcaaaaaagg 11760gaataagggc gacacggaaa tgttgaatac
tcatactctt cctttttcaa tattattgaa 11820gcatttatca gggttattgt
ctcatgagcg gatacatatt tgaatgtatt tagaaaaata 11880aacaaatagg
ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca
11940ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
cgtcttcaag 12000aattcat 120072211893DNAArtificial SequenceRCR
Vector - pAC3-yCD2 22tagttattaa tagtaatcaa ttacggggtc attagttcat
agcccatata tggagttccg 60cgttacataa cttacggtaa atggcccgcc tggctgaccg
cccaacgacc cccgcccatt 120gacgtcaata atgacgtatg ttcccatagt
aacgccaata gggactttcc attgacgtca 180atgggtggag tatttacggt
aaactgccca cttggcagta catcaagtgt atcatatgcc 240aagtacgccc
cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
300catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
tcgctattac 360catggtgatg cggttttggc agtacatcaa tgggcgtgga
tagcggtttg actcacgggg 420atttccaagt ctccacccca ttgacgtcaa
tgggagtttg ttttggcacc aaaatcaacg 480ggactttcca aaatgtcgta
acaactccgc cccattgacg caaatgggcg gtaggcgtgt 540acggtgggag
gtctatataa gcagagctgg tttagtgaac cggcgccagt cctccgattg
600actgagtcgc ccgggtaccc gtgtatccaa taaaccctct tgcagttgca
tccgacttgt 660ggtctcgctg ttccttggga gggtctcctc tgagtgattg
actacccgtc agcgggggtc 720tttcatttgg gggctcgtcc gggatcggga
gacccctgcc cagggaccac cgacccacca 780ccgggaggta agctggccag
caacttatct gtgtctgtcc gattgtctag tgtctatgac 840tgattttatg
cgcctgcgtc ggtactagtt agctaactag ctctgtatct ggcggacccg
900tggtggaact gacgagttcg gaacacccgg ccgcaaccct gggagacgtc
ccagggactt 960cgggggccgt ttttgtggcc cgacctgagt ccaaaaatcc
cgatcgtttt ggactctttg 1020gtgcaccccc cttagaggag ggatatgtgg
ttctggtagg agacgagaac ctaaaacagt 1080tcccgcctcc gtctgaattt
ttgctttcgg tttgggaccg aagccgcgcc gcgcgtcttg 1140tctgctgcag
catcgttctg tgttgtctct gtctgactgt gtttctgtat ttgtctgaaa
1200atatgggcca gactgttacc actcccttaa gtttgacctt aggtcactgg
aaagatgtcg 1260agcggatcgc tcacaaccag tcggtagatg tcaagaagag
acgttgggtt accttctgct 1320ctgcagaatg gccaaccttt aacgtcggat
ggccgcgaga cggcaccttt aaccgagacc 1380tcatcaccca ggttaagatc
aaggtctttt cacctggccc gcatggacac ccagaccagg 1440tcccctacat
cgtgacctgg gaagccttgg cttttgaccc ccctccctgg gtcaagccct
1500ttgtacaccc taagcctccg cctcctcttc ctccatccgc cccgtctctc
ccccttgaac 1560ctcctcgttc gaccccgcct cgatcctccc tttatccagc
cctcactcct tctctaggcg 1620ccaaacctaa acctcaagtt ctttctgaca
gtggggggcc gctcatcgac ctacttacag 1680aagacccccc gccttatagg
gacccaagac cacccccttc cgacagggac ggaaatggtg 1740gagaagcgac
ccctgcggga gaggcaccgg acccctcccc aatggcatct cgcctacgtg
1800ggagacggga gccccctgtg gccgactcca ctacctcgca ggcattcccc
ctccgcgcag 1860gaggaaacgg acagcttcaa tactggccgt tctcctcttc
tgacctttac aactggaaaa 1920ataataaccc ttctttttct gaagatccag
gtaaactgac agctctgatc gagtctgtcc 1980tcatcaccca tcagcccacc
tgggacgact gtcagcagct gttggggact ctgctgaccg 2040gagaagaaaa
acaacgggtg ctcttagagg ctagaaaggc ggtgcggggc gatgatgggc
2100gccccactca actgcccaat gaagtcgatg ccgcttttcc cctcgagcgc
ccagactggg 2160attacaccac ccaggcaggt aggaaccacc tagtccacta
tcgccagttg ctcctagcgg 2220gtctccaaaa cgcgggcaga agccccacca
atttggccaa ggtaaaagga ataacacaag 2280ggcccaatga gtctccctcg
gccttcctag agagacttaa ggaagcctat cgcaggtaca 2340ctccttatga
ccctgaggac ccagggcaag aaactaatgt gtctatgtct ttcatttggc
2400agtctgcccc agacattggg agaaagttag agaggttaga agatttaaaa
aacaagacgc 2460ttggagattt ggttagagag gcagaaaaga tctttaataa
acgagaaacc ccggaagaaa 2520gagaggaacg tatcaggaga gaaacagagg
aaaaagaaga acgccgtagg acagaggatg 2580agcagaaaga gaaagaaaga
gatcgtagga gacatagaga gatgagcaag ctattggcca 2640ctgtcgttag
tggacagaaa caggatagac agggaggaga acgaaggagg tcccaactcg
2700atcgcgacca gtgtgcctac tgcaaagaaa aggggcactg ggctaaagat
tgtcccaaga 2760aaccacgagg acctcgggga ccaagacccc agacctccct
cctgacccta gatgactagg 2820gaggtcaggg tcaggagccc ccccctgaac
ccaggataac cctcaaagtc ggggggcaac 2880ccgtcacctt cctggtagat
actggggccc aacactccgt gctgacccaa aatcctggac 2940ccctaagtga
taagtctgcc tgggtccaag gggctactgg aggaaagcgg tatcgctgga
3000ccacggatcg caaagtacat ctagctaccg gtaaggtcac ccactctttc
ctccatgtac 3060cagactgtcc ctatcctctg ttaggaagag atttgctgac
taaactaaaa gcccaaatcc 3120actttgaggg atcaggagcc caggttatgg
gaccaatggg gcagcccctg caagtgttga 3180ccctaaatat agaagatgag
tatcggctac atgagacctc aaaagagcca gatgtttctc 3240tagggtccac
atggctgtct gattttcctc aggcctgggc ggaaaccggg ggcatgggac
3300tggcagttcg ccaagctcct ctgatcatac ctctgaaagc aacctctacc
cccgtgtcca 3360taaaacaata ccccatgtca caagaagcca gactggggat
caagccccac atacagagac 3420tgttggacca gggaatactg gtaccctgcc
agtccccctg gaacacgccc ctgctacccg 3480ttaagaaacc agggactaat
gattataggc ctgtccagga tctgagagaa gtcaacaagc 3540gggtggaaga
catccacccc accgtgccca acccttacaa cctcttgagc gggctcccac
3600cgtcccacca gtggtacact gtgcttgatt taaaggatgc ctttttctgc
ctgagactcc 3660accccaccag tcagcctctc ttcgcctttg agtggagaga
tccagagatg ggaatctcag 3720gacaattgac ctggaccaga ctcccacagg
gtttcaaaaa cagtcccacc ctgtttgatg 3780aggcactgca cagagaccta
gcagacttcc ggatccagca cccagacttg atcctgctac 3840agtacgtgga
tgacttactg ctggccgcca cttctgagct agactgccaa caaggtactc
3900gggccctgtt acaaacccta gggaacctcg ggtatcgggc ctcggccaag
aaagcccaaa 3960tttgccagaa acaggtcaag tatctggggt atcttctaaa
agagggtcag agatggctga 4020ctgaggccag aaaagagact gtgatggggc
agcctactcc gaagacccct cgacaactaa 4080gggagttcct agggacggca
ggcttctgtc gcctctggat ccctgggttt gcagaaatgg 4140cagccccctt
gtaccctctc accaaaacgg ggactctgtt taattggggc ccagaccaac
4200aaaaggccta tcaagaaatc aagcaagctc ttctaactgc cccagccctg
gggttgccag 4260atttgactaa gccctttgaa ctctttgtcg acgagaagca
gggctacgcc aaaggtgtcc 4320taacgcaaaa actgggacct tggcgtcggc
cggtggccta cctgtccaaa aagctagacc 4380cagtagcagc tgggtggccc
ccttgcctac ggatggtagc agccattgcc gtactgacaa 4440aggatgcagg
caagctaacc atgggacagc cactagtcat tctggccccc catgcagtag
4500aggcactagt caaacaaccc cccgaccgct ggctttccaa cgcccggatg
actcactatc 4560aggccttgct tttggacacg gaccgggtcc agttcggacc
ggtggtagcc ctgaacccgg 4620ctacgctgct cccactgcct gaggaagggc
tgcaacacaa ctgccttgat atcctggccg 4680aagcccacgg aacccgaccc
gacctaacgg accagccgct cccagacgcc gaccacacct 4740ggtacacgga
tggaagcagt ctcttacaag agggacagcg taaggcggga gctgcggtga
4800ccaccgagac cgaggtaatc tgggctaaag ccctgccagc cgggacatcc
gctcagcggg 4860ctgaactgat agcactcacc caggccctaa agatggcaga
aggtaagaag ctaaatgttt 4920atactgatag ccgttatgct tttgctactg
cccatatcca tggagaaata tacagaaggc 4980gtgggttgct cacatcagaa
ggcaaagaga tcaaaaataa agacgagatc ttggccctac 5040taaaagccct
ctttctgccc aaaagactta gcataatcca ttgtccagga catcaaaagg
5100gacacagcgc cgaggctaga ggcaaccgga tggctgacca agcggcccga
aaggcagcca 5160tcacagagac tccagacacc tctaccctcc tcatagaaaa
ttcatcaccc tacacctcag 5220aacattttca ttacacagtg actgatataa
aggacctaac caagttgggg gccatttatg 5280ataaaacaaa gaagtattgg
gtctaccaag gaaaacctgt gatgcctgac cagtttactt 5340ttgaattatt
agactttctt catcagctga ctcacctcag cttctcaaaa atgaaggctc
5400tcctagagag aagccacagt ccctactaca tgctgaaccg ggatcgaaca
ctcaaaaata 5460tcactgagac ctgcaaagct tgtgcacaag tcaacgccag
caagtctgcc gttaaacagg 5520gaactagggt ccgcgggcat cggcccggca
ctcattggga gatcgatttc accgagataa 5580agcccggatt gtatggctat
aaatatcttc tagtttttat agataccttt tctggctgga 5640tagaagcctt
cccaaccaag aaagaaaccg ccaaggtcgt aaccaagaag ctactagagg
5700agatcttccc caggttcggc atgcctcagg tattgggaac tgacaatggg
cctgccttcg 5760tctccaaggt gagtcagaca gtggccgatc tgttggggat
tgattggaaa ttacattgtg 5820catacagacc ccaaagctca ggccaggtag
aaagaatgaa tagaaccatc aaggagactt 5880taactaaatt aacgcttgca
actggctcta gagactgggt gctcctactc cccttagccc 5940tgtaccgagc
ccgcaacacg ccgggccccc atggcctcac cccatatgag atcttatatg
6000gggcaccccc gccccttgta aacttccctg accctgacat gacaagagtt
actaacagcc 6060cctctctcca agctcactta caggctctct acttagtcca
gcacgaagtc tggagacctc 6120tggcggcagc ctaccaagaa caactggacc
gaccggtggt acctcaccct taccgagtcg 6180gcgacacagt gtgggtccgc
cgacaccaga ctaagaacct agaacctcgc tggaaaggac 6240cttacacagt
cctgctgacc acccccaccg ccctcaaagt agacggcatc gcagcttgga
6300tacacgccgc ccacgtgaag gctgccgacc ccgggggtgg accatcctct
agactgacat 6360ggcgcgttca acgctctcaa aaccccctca agataagatt
aacccgtgga agcccttaat 6420agtcatggga gtcctgttag gagtagggat
ggcagagagc ccccatcagg tctttaatgt 6480aacctggaga gtcaccaacc
tgatgactgg gcgtaccgcc aatgccacct ccctcctggg 6540aactgtacaa
gatgccttcc caaaattata ttttgatcta tgtgatctgg tcggagagga
6600gtgggaccct tcagaccagg aaccgtatgt cgggtatggc tgcaagtacc
ccgcagggag 6660acagcggacc cggacttttg acttttacgt gtgccctggg
cataccgtaa agtcggggtg 6720tgggggacca ggagagggct actgtggtaa
atgggggtgt gaaaccaccg gacaggctta 6780ctggaagccc acatcatcgt
gggacctaat ctcccttaag cgcggtaaca ccccctggga 6840cacgggatgc
tctaaagttg cctgtggccc ctgctacgac ctctccaaag tatccaattc
6900cttccaaggg gctactcgag ggggcagatg caaccctcta gtcctagaat
tcactgatgc 6960aggaaaaaag gctaactggg acgggcccaa atcgtgggga
ctgagactgt accggacagg 7020aacagatcct attaccatgt tctccctgac
ccggcaggtc cttaatgtgg gaccccgagt 7080ccccataggg cccaacccag
tattacccga ccaaagactc ccttcctcac caatagagat 7140tgtaccggct
ccacagccac ctagccccct caataccagt tacccccctt ccactaccag
7200tacaccctca acctccccta caagtccaag tgtcccacag ccacccccag
gaactggaga 7260tagactacta gctctagtca aaggagccta tcaggcgctt
aacctcacca atcccgacaa 7320gacccaagaa tgttggctgt gcttagtgtc
gggacctcct tattacgaag gagtagcggt 7380cgtgggcact tataccaatc
attccaccgc tccggccaac tgtacggcca cttcccaaca 7440taagcttacc
ctatctgaag tgacaggaca gggcctatgc atgggggcag tacctaaaac
7500tcaccaggcc ttatgtaaca ccacccaaag cgccggctca ggatcctact
accttgcagc 7560acccgccgga acaatgtggg cttgcagcac tggattgact
ccctgcttgt ccaccacggt 7620gctcaatcta accacagatt attgtgtatt
agttgaactc tggcccagag taatttacca 7680ctcccccgat tatatgtatg
gtcagcttga acagcgtacc aaatataaaa gagagccagt 7740atcattgacc
ctggcccttc tactaggagg attaaccatg ggagggattg cagctggaat
7800agggacgggg accactgcct taattaaaac ccagcagttt gagcagcttc
atgccgctat 7860ccagacagac ctcaacgaag tcgaaaagtc aattaccaac
ctagaaaagt cactgacctc 7920gttgtctgaa gtagtcctac agaaccgcag
aggcctagat ttgctattcc taaaggaggg 7980aggtctctgc gcagccctaa
aagaagaatg ttgtttttat gcagaccaca cggggctagt 8040gagagacagc
atggccaaat taagagaaag gcttaatcag agacaaaaac tatttgagac
8100aggccaagga tggttcgaag ggctgtttaa tagatccccc tggtttacca
ccttaatctc 8160caccatcatg ggacctctaa tagtactctt actgatctta
ctctttggac cttgcattct 8220caatcgattg gtccaatttg ttaaagacag
gatctcagtg gtccaggctc tggttttgac 8280tcagcaatat caccagctaa
aacccataga gtacgagcca tgaacgcgtt actggccgaa 8340gccgcttgga
ataaggccgg tgtgcgtttg tctatatgtt attttccacc atattgccgt
8400cttttggcaa tgtgagggcc cggaaacctg gccctgtctt cttgacgagc
attcctaggg 8460gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa
tgtcgtgaag gaagcagttc 8520ctctggaagc ttcttgaaga caaacaacgt
ctgtagcgac cctttgcagg cagcggaacc 8580ccccacctgg cgacaggtgc
ctctgcggcc aaaagccacg tgtataagat acacctgcaa 8640aggcggcaca
accccagtgc cacgttgtga gttggatagt tgtggaaaga gtcaaatggc
8700tctcctcaag cgtattcaac aaggggctga aggatgccca gaaggtaccc
cattgtatgg 8760gatctgatct ggggcctcgg tgcacatgct ttacatgtgt
ttagtcgagg ttaaaaaaac 8820gtctaggccc cccgaaccac ggggacgtgg
ttttcctttg aaaaacacga ttataaatgg 8880tgaccggcgg catggcctcc
aagtgggatc aaaagggcat ggatatcgct tacgaggagg 8940ccctgctggg
ctacaaggag ggcggcgtgc ctatcggcgg ctgtctgatc aacaacaagg
9000acggcagtgt gctgggcagg ggccacaaca tgaggttcca gaagggctcc
gccaccctgc 9060acggcgagat ctccaccctg gagaactgtg gcaggctgga
gggcaaggtg tacaaggaca 9120ccaccctgta caccaccctg tccccttgtg
acatgtgtac cggcgctatc atcatgtacg 9180gcatccctag gtgtgtgatc
ggcgagaacg tgaacttcaa gtccaagggc gagaagtacc 9240tgcaaaccag
gggccacgag gtggtggttg ttgacgatga gaggtgtaag aagctgatga
9300agcagttcat cgacgagagg cctcaggact ggttcgagga tatcggcgag
taagcggccg 9360cagataaaat aaaagatttt atttagtctc cagaaaaagg
ggggaatgaa agaccccacc 9420tgtaggtttg gcaagctagc ttaagtaacg
ccattttgca aggcatggaa aaatacataa 9480ctgagaatag agaagttcag
atcaaggtca ggaacagatg gaacagctga atatgggcca 9540aacaggatat
ctgtggtaag cagttcctgc cccggctcag ggccaagaac agatggaaca
9600gctgaatatg ggccaaacag gatatctgtg gtaagcagtt cctgccccgg
ctcagggcca 9660agaacagatg gtccccagat gcggtccagc cctcagcagt
ttctagagaa ccatcagatg 9720tttccagggt gccccaagga cctgaaatga
ccctgtgcct tatttgaact aaccaatcag 9780ttcgcttctc gcttctgttc
gcgcgcttct gctccccgag ctcaataaaa gagcccacaa 9840cccctcactc
ggggcgccag tcctccgatt gactgagtcg cccgggtacc cgtgtatcca
9900ataaaccctc ttgcagttgc atccgacttg tggtctcgct gttccttggg
agggtctcct 9960ctgagtgatt gactacccgt cagcgggggt ctttcattac
atgtgagcaa aaggccagca 10020aaaggccagg aaccgtaaaa aggccgcgtt
gctggcgttt ttccataggc tccgcccccc 10080tgacgagcat cacaaaaatc
gacgctcaag tcagaggtgg cgaaacccga caggactata 10140aagataccag
gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
10200gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
ctcatagctc 10260acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
aagctgggct gtgtgcacga 10320accccccgtt cagcccgacc gctgcgcctt
atccggtaac tatcgtcttg agtccaaccc 10380ggtaagacac gacttatcgc
cactggcagc agccactggt aacaggatta gcagagcgag 10440gtatgtaggc
ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
10500gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
gagttggtag 10560ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
ttttttgttt gcaagcagca 10620gattacgcgc agaaaaaaag gatctcaaga
agatcctttg atcttttcta cggggtctga 10680cgctcagtgg aacgaaaact
cacgttaagg gattttggtc atgagattat caaaaaggat 10740cttcacctag
atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
10800gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
cagcgatctg 10860tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
tagataacta cgatacggga 10920gggcttacca tctggcccca gtgctgcaat
gataccgcga gacccacgct caccggctcc 10980agatttatca gcaataaacc
agccagccgg aagggccgag cgcagaagtg gtcctgcaac 11040tttatccgcc
tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
11100agttaatagt ttgcgcaacg ttgttgccat tgctgcaggc atcgtggtgt
cacgctcgtc 11160gtttggtatg gcttcattca gctccggttc ccaacgatca
aggcgagtta catgatcccc 11220catgttgtgc aaaaaagcgg ttagctcctt
cggtcctccg atcgttgtca gaagtaagtt 11280ggccgcagtg ttatcactca
tggttatggc agcactgcat aattctctta ctgtcatgcc 11340atccgtaaga
tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
11400tatgcggcga ccgagttgct cttgcccggc gtcaacacgg gataataccg
cgccacatag 11460cagaacttta aaagtgctca tcattggaaa acgttcttcg
gggcgaaaac tctcaaggat 11520cttaccgctg ttgagatcca gttcgatgta
acccactcgt gcacccaact gatcttcagc 11580atcttttact ttcaccagcg
tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa 11640aaagggaata
agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
11700ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
gtatttagaa 11760aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
gtgccacctg acgtctaaga 11820aaccattatt atcatgacat taacctataa
aaataggcgt atcacgaggc cctttcgtct 11880tcaagaattc cat
11893234473DNAHomo sapiensCDS(175)..(3942) 23aaggggaggt aaccctggcc
cctttggtcg gggccccggg cagccgcgcg ccccttccca 60cggggccctt tactgcgccg
cgcgcccggc ccccacccct cgcagcaccc cgcgccccgc 120gccctcccag
ccgggtccag ccggagccat ggggccggag ccgcagtgag cacc atg 177 Met 1 gag
ctg gcg gcc ttg tgc cgc tgg ggg ctc ctc ctc gcc ctc ttg ccc 225Glu
Leu Ala Ala Leu Cys Arg Trp Gly Leu Leu Leu Ala Leu Leu Pro 5 10 15
ccc gga gcc gcg agc acc caa gtg tgc acc ggc aca gac atg aag ctg
273Pro Gly Ala Ala Ser Thr Gln Val Cys Thr Gly Thr Asp Met Lys Leu
20 25 30 cgg ctc cct gcc agt ccc gag acc cac ctg gac atg ctc cgc
cac ctc 321Arg Leu Pro Ala Ser Pro Glu Thr His Leu Asp Met Leu Arg
His Leu 35 40 45 tac cag ggc tgc cag gtg gtg cag gga aac ctg gaa
ctc acc tac ctg 369Tyr Gln Gly Cys Gln Val Val Gln Gly Asn Leu Glu
Leu Thr Tyr Leu 50 55 60 65 ccc acc aat gcc agc ctg tcc ttc ctg cag
gat atc cag gag gtg cag 417Pro Thr Asn Ala Ser Leu Ser Phe Leu Gln
Asp Ile Gln Glu Val Gln 70 75 80 ggc tac gtg ctc atc gct cac aac
caa gtg agg cag gtc cca ctg cag 465Gly Tyr Val Leu Ile Ala His Asn
Gln Val Arg Gln Val Pro Leu Gln 85 90 95 agg ctg cgg att gtg cga
ggc acc cag ctc ttt gag gac aac tat gcc 513Arg Leu Arg Ile Val Arg
Gly Thr Gln Leu Phe Glu Asp Asn Tyr Ala 100 105 110 ctg gcc gtg cta
gac aat gga gac ccg ctg aac aat acc acc cct gtc 561Leu Ala Val Leu
Asp Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro Val 115 120 125 aca ggg
gcc tcc cca gga ggc ctg cgg gag ctg cag ctt cga agc ctc 609Thr Gly
Ala Ser Pro Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser Leu 130 135 140
145 aca gag atc ttg aaa gga ggg gtc ttg atc cag cgg aac ccc cag ctc
657Thr Glu Ile Leu Lys Gly Gly Val Leu Ile Gln Arg Asn Pro Gln Leu
150 155 160 tgc tac cag gac acg att ttg tgg aag gac atc ttc cac aag
aac aac 705Cys Tyr Gln Asp Thr Ile Leu Trp Lys Asp Ile Phe His Lys
Asn Asn 165 170 175 cag ctg gct ctc aca ctg ata gac acc aac cgc tct
cgg gcc tgc cac 753Gln Leu Ala Leu Thr Leu Ile Asp Thr Asn Arg Ser
Arg Ala Cys His 180 185 190 ccc tgt tct ccg atg tgt aag ggc tcc cgc
tgc tgg gga gag agt tct 801Pro Cys Ser Pro Met Cys Lys Gly Ser Arg
Cys Trp Gly Glu Ser Ser 195 200 205 gag gat tgt cag agc ctg acg cgc
act gtc tgt gcc ggt ggc tgt gcc 849Glu Asp Cys Gln Ser Leu Thr Arg
Thr Val Cys Ala Gly Gly Cys Ala 210 215 220 225 cgc tgc aag ggg cca
ctg ccc act gac tgc tgc cat gag cag tgt gct 897Arg Cys Lys Gly Pro
Leu Pro Thr Asp Cys Cys His Glu Gln Cys Ala 230 235 240 gcc ggc tgc
acg ggc ccc aag cac tct gac tgc ctg gcc tgc ctc cac 945Ala Gly Cys
Thr Gly Pro Lys His Ser Asp Cys Leu Ala Cys Leu His 245 250 255 ttc
aac cac agt ggc atc tgt gag ctg cac tgc cca gcc ctg gtc acc 993Phe
Asn His Ser Gly Ile Cys Glu Leu His Cys Pro Ala Leu Val Thr 260 265
270 tac aac aca gac acg ttt gag tcc atg ccc aat ccc gag ggc cgg tat
1041Tyr Asn Thr Asp Thr Phe Glu Ser Met Pro Asn Pro Glu Gly Arg Tyr
275 280 285 aca ttc ggc gcc agc tgt gtg act gcc tgt ccc tac aac tac
ctt tct 1089Thr Phe Gly Ala Ser Cys Val Thr Ala Cys Pro Tyr Asn Tyr
Leu Ser 290 295 300 305 acg gac gtg gga tcc tgc acc ctc gtc tgc ccc
ctg cac aac caa gag 1137Thr Asp Val Gly Ser Cys Thr Leu Val Cys Pro
Leu His Asn Gln Glu 310 315 320 gtg aca gca gag gat gga aca cag cgg
tgt gag aag tgc agc aag ccc 1185Val Thr Ala Glu Asp Gly Thr Gln Arg
Cys Glu Lys Cys Ser Lys Pro 325 330 335 tgt gcc cga gtg tgc tat ggt
ctg ggc atg gag cac ttg cga gag gtg 1233Cys Ala Arg Val Cys Tyr Gly
Leu Gly Met Glu His Leu Arg Glu Val 340 345 350 agg gca gtt acc agt
gcc aat atc cag gag ttt gct ggc tgc aag aag 1281Arg Ala Val Thr Ser
Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys Lys 355 360 365 atc ttt ggg
agc ctg gca ttt ctg ccg gag agc ttt gat ggg gac cca 1329Ile Phe Gly
Ser Leu Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp Pro 370 375 380 385
gcc tcc aac act gcc ccg ctc cag cca gag cag ctc caa gtg ttt gag
1377Ala Ser Asn Thr Ala Pro Leu Gln Pro Glu Gln Leu Gln Val Phe Glu
390 395 400 act ctg gaa gag atc aca ggt tac cta tac atc tca gca tgg
ccg gac 1425Thr Leu Glu Glu Ile Thr Gly Tyr Leu Tyr Ile Ser Ala Trp
Pro Asp 405 410 415 agc ctg cct gac ctc agc gtc ttc cag aac ctg caa
gta atc cgg gga 1473Ser Leu Pro Asp Leu Ser Val Phe Gln Asn Leu Gln
Val Ile Arg Gly 420 425 430 cga att ctg cac aat ggc gcc tac tcg ctg
acc ctg caa ggg ctg ggc 1521Arg Ile Leu His Asn Gly Ala Tyr Ser Leu
Thr Leu Gln Gly Leu Gly 435 440 445 atc agc tgg ctg ggg ctg cgc tca
ctg agg gaa ctg ggc agt gga ctg 1569Ile Ser Trp Leu Gly Leu Arg Ser
Leu Arg
Glu Leu Gly Ser Gly Leu 450 455 460 465 gcc ctc atc cac cat aac acc
cac ctc tgc ttc gtg cac acg gtg ccc 1617Ala Leu Ile His His Asn Thr
His Leu Cys Phe Val His Thr Val Pro 470 475 480 tgg gac cag ctc ttt
cgg aac ccg cac caa gct ctg ctc cac act gcc 1665Trp Asp Gln Leu Phe
Arg Asn Pro His Gln Ala Leu Leu His Thr Ala 485 490 495 aac cgg cca
gag gac gag tgt gtg ggc gag ggc ctg gcc tgc cac cag 1713Asn Arg Pro
Glu Asp Glu Cys Val Gly Glu Gly Leu Ala Cys His Gln 500 505 510 ctg
tgc gcc cga ggg cac tgc tgg ggt cca ggg ccc acc cag tgt gtc 1761Leu
Cys Ala Arg Gly His Cys Trp Gly Pro Gly Pro Thr Gln Cys Val 515 520
525 aac tgc agc cag ttc ctt cgg ggc cag gag tgc gtg gag gaa tgc cga
1809Asn Cys Ser Gln Phe Leu Arg Gly Gln Glu Cys Val Glu Glu Cys Arg
530 535 540 545 gta ctg cag ggg ctc ccc agg gag tat gtg aat gcc agg
cac tgt ttg 1857Val Leu Gln Gly Leu Pro Arg Glu Tyr Val Asn Ala Arg
His Cys Leu 550 555 560 ccg tgc cac cct gag tgt cag ccc cag aat ggc
tca gtg acc tgt ttt 1905Pro Cys His Pro Glu Cys Gln Pro Gln Asn Gly
Ser Val Thr Cys Phe 565 570 575 gga ccg gag gct gac cag tgt gtg gcc
tgt gcc cac tat aag gac cct 1953Gly Pro Glu Ala Asp Gln Cys Val Ala
Cys Ala His Tyr Lys Asp Pro 580 585 590 ccc ttc tgc gtg gcc cgc tgc
ccc agc ggt gtg aaa cct gac ctc tcc 2001Pro Phe Cys Val Ala Arg Cys
Pro Ser Gly Val Lys Pro Asp Leu Ser 595 600 605 tac atg ccc atc tgg
aag ttt cca gat gag gag ggc gca tgc cag cct 2049Tyr Met Pro Ile Trp
Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln Pro 610 615 620 625 tgc ccc
atc aac tgc acc cac tcc tgt gtg gac ctg gat gac aag ggc 2097Cys Pro
Ile Asn Cys Thr His Ser Cys Val Asp Leu Asp Asp Lys Gly 630 635 640
tgc ccc gcc gag cag aga gcc agc cct ctg acg tcc atc atc tct gcg
2145Cys Pro Ala Glu Gln Arg Ala Ser Pro Leu Thr Ser Ile Ile Ser Ala
645 650 655 gtg gtt ggc att ctg ctg gtc gtg gtc ttg ggg gtg gtc ttt
ggg atc 2193Val Val Gly Ile Leu Leu Val Val Val Leu Gly Val Val Phe
Gly Ile 660 665 670 ctc atc aag cga cgg cag cag aag atc cgg aag tac
acg atg cgg aga 2241Leu Ile Lys Arg Arg Gln Gln Lys Ile Arg Lys Tyr
Thr Met Arg Arg 675 680 685 ctg ctg cag gaa acg gag ctg gtg gag ccg
ctg aca cct agc gga gcg 2289Leu Leu Gln Glu Thr Glu Leu Val Glu Pro
Leu Thr Pro Ser Gly Ala 690 695 700 705 atg ccc aac cag gcg cag atg
cgg atc ctg aaa gag acg gag ctg agg 2337Met Pro Asn Gln Ala Gln Met
Arg Ile Leu Lys Glu Thr Glu Leu Arg 710 715 720 aag gtg aag gtg ctt
gga tct ggc gct ttt ggc aca gtc tac aag ggc 2385Lys Val Lys Val Leu
Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys Gly 725 730 735 atc tgg atc
cct gat ggg gag aat gtg aaa att cca gtg gcc atc aaa 2433Ile Trp Ile
Pro Asp Gly Glu Asn Val Lys Ile Pro Val Ala Ile Lys 740 745 750 gtg
ttg agg gaa aac aca tcc ccc aaa gcc aac aaa gaa atc tta gac 2481Val
Leu Arg Glu Asn Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu Asp 755 760
765 gaa gca tac gtg atg gct ggt gtg ggc tcc cca tat gtc tcc cgc ctt
2529Glu Ala Tyr Val Met Ala Gly Val Gly Ser Pro Tyr Val Ser Arg Leu
770 775 780 785 ctg ggc atc tgc ctg aca tcc acg gtg cag ctg gtg aca
cag ctt atg 2577Leu Gly Ile Cys Leu Thr Ser Thr Val Gln Leu Val Thr
Gln Leu Met 790 795 800 ccc tat ggc tgc ctc tta gac cat gtc cgg gaa
aac cgc gga cgc ctg 2625Pro Tyr Gly Cys Leu Leu Asp His Val Arg Glu
Asn Arg Gly Arg Leu 805 810 815 ggc tcc cag gac ctg ctg aac tgg tgt
atg cag att gcc aag ggg atg 2673Gly Ser Gln Asp Leu Leu Asn Trp Cys
Met Gln Ile Ala Lys Gly Met 820 825 830 agc tac ctg gag gat gtg cgg
ctc gta cac agg gac ttg gcc gct cgg 2721Ser Tyr Leu Glu Asp Val Arg
Leu Val His Arg Asp Leu Ala Ala Arg 835 840 845 aac gtg ctg gtc aag
agt ccc aac cat gtc aaa att aca gac ttc ggg 2769Asn Val Leu Val Lys
Ser Pro Asn His Val Lys Ile Thr Asp Phe Gly 850 855 860 865 ctg gct
cgg ctg ctg gac att gac gag aca gag tac cat gca gat ggg 2817Leu Ala
Arg Leu Leu Asp Ile Asp Glu Thr Glu Tyr His Ala Asp Gly 870 875 880
ggc aag gtg ccc atc aag tgg atg gcg ctg gag tcc att ctc cgc cgg
2865Gly Lys Val Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu Arg Arg
885 890 895 cgg ttc acc cac cag agt gat gtg tgg agt tat ggt gtg act
gtg tgg 2913Arg Phe Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr
Val Trp 900 905 910 gag ctg atg act ttt ggg gcc aaa cct tac gat ggg
atc cca gcc cgg 2961Glu Leu Met Thr Phe Gly Ala Lys Pro Tyr Asp Gly
Ile Pro Ala Arg 915 920 925 gag atc cct gac ctg ctg gaa aag ggg gag
cgg ctg ccc cag ccc ccc 3009Glu Ile Pro Asp Leu Leu Glu Lys Gly Glu
Arg Leu Pro Gln Pro Pro 930 935 940 945 atc tgc acc att gat gtc tac
atg atc atg gtc aaa tgt tgg atg att 3057Ile Cys Thr Ile Asp Val Tyr
Met Ile Met Val Lys Cys Trp Met Ile 950 955 960 gac tct gaa tgt cgg
cca aga ttc cgg gag ttg gtg tct gaa ttc tcc 3105Asp Ser Glu Cys Arg
Pro Arg Phe Arg Glu Leu Val Ser Glu Phe Ser 965 970 975 cgc atg gcc
agg gac ccc cag cgc ttt gtg gtc atc cag aat gag gac 3153Arg Met Ala
Arg Asp Pro Gln Arg Phe Val Val Ile Gln Asn Glu Asp 980 985 990 ttg
ggc cca gcc agt ccc ttg gac agc acc ttc tac cgc tca ctg ctg 3201Leu
Gly Pro Ala Ser Pro Leu Asp Ser Thr Phe Tyr Arg Ser Leu Leu 995
1000 1005 gag gac gat gac atg ggg gac ctg gtg gat gct gag gag tat
ctg 3246Glu Asp Asp Asp Met Gly Asp Leu Val Asp Ala Glu Glu Tyr Leu
1010 1015 1020 gta ccc cag cag ggc ttc ttc tgt cca gac cct gcc ccg
ggc gct 3291Val Pro Gln Gln Gly Phe Phe Cys Pro Asp Pro Ala Pro Gly
Ala 1025 1030 1035 ggg ggc atg gtc cac cac agg cac cgc agc tca tct
acc agg agt 3336Gly Gly Met Val His His Arg His Arg Ser Ser Ser Thr
Arg Ser 1040 1045 1050 ggc ggt ggg gac ctg aca cta ggg ctg gag ccc
tct gaa gag gag 3381Gly Gly Gly Asp Leu Thr Leu Gly Leu Glu Pro Ser
Glu Glu Glu 1055 1060 1065 gcc ccc agg tct cca ctg gca ccc tcc gaa
ggg gct ggc tcc gat 3426Ala Pro Arg Ser Pro Leu Ala Pro Ser Glu Gly
Ala Gly Ser Asp 1070 1075 1080 gta ttt gat ggt gac ctg gga atg ggg
gca gcc aag ggg ctg caa 3471Val Phe Asp Gly Asp Leu Gly Met Gly Ala
Ala Lys Gly Leu Gln 1085 1090 1095 agc ctc ccc aca cat gac ccc agc
cct cta cag cgg tac agt gag 3516Ser Leu Pro Thr His Asp Pro Ser Pro
Leu Gln Arg Tyr Ser Glu 1100 1105 1110 gac ccc aca gta ccc ctg ccc
tct gag act gat ggc tac gtt gcc 3561Asp Pro Thr Val Pro Leu Pro Ser
Glu Thr Asp Gly Tyr Val Ala 1115 1120 1125 ccc ctg acc tgc agc ccc
cag cct gaa tat gtg aac cag cca gat 3606Pro Leu Thr Cys Ser Pro Gln
Pro Glu Tyr Val Asn Gln Pro Asp 1130 1135 1140 gtt cgg ccc cag ccc
cct tcg ccc cga gag ggc cct ctg cct gct 3651Val Arg Pro Gln Pro Pro
Ser Pro Arg Glu Gly Pro Leu Pro Ala 1145 1150 1155 gcc cga cct gct
ggt gcc act ctg gaa agg ccc aag act ctc tcc 3696Ala Arg Pro Ala Gly
Ala Thr Leu Glu Arg Pro Lys Thr Leu Ser 1160 1165 1170 cca ggg aag
aat ggg gtc gtc aaa gac gtt ttt gcc ttt ggg ggt 3741Pro Gly Lys Asn
Gly Val Val Lys Asp Val Phe Ala Phe Gly Gly 1175 1180 1185 gcc gtg
gag aac ccc gag tac ttg aca ccc cag gga gga gct gcc 3786Ala Val Glu
Asn Pro Glu Tyr Leu Thr Pro Gln Gly Gly Ala Ala 1190 1195 1200 cct
cag ccc cac cct cct cct gcc ttc agc cca gcc ttc gac aac 3831Pro Gln
Pro His Pro Pro Pro Ala Phe Ser Pro Ala Phe Asp Asn 1205 1210 1215
ctc tat tac tgg gac cag gac cca cca gag cgg ggg gct cca ccc 3876Leu
Tyr Tyr Trp Asp Gln Asp Pro Pro Glu Arg Gly Ala Pro Pro 1220 1225
1230 agc acc ttc aaa ggg aca cct acg gca gag aac cca gag tac ctg
3921Ser Thr Phe Lys Gly Thr Pro Thr Ala Glu Asn Pro Glu Tyr Leu
1235 1240 1245 ggt ctg gac gtg cca gtg tga accagaaggc caagtccgca
gaagccctga 3972Gly Leu Asp Val Pro Val 1250 1255 tgtgtcctca
gggagcaggg aaggcctgac ttctgctggc atcaagaggt gggagggccc
4032tccgaccact tccaggggaa cctgccatgc caggaacctg tcctaaggaa
ccttccttcc 4092tgcttgagtt cccagatggc tggaaggggt ccagcctcgt
tggaagagga acagcactgg 4152ggagtctttg tggattctga ggccctgccc
aatgagactc tagggtccag tggatgccac 4212agcccagctt ggccctttcc
ttccagatcc tgggtactga aagccttagg gaagctggcc 4272tgagagggga
agcggcccta agggagtgtc taagaacaaa agcgacccat tcagagactg
4332tccctgaaac ctagtactgc cccccatgag gaaggaacag caatggtgtc
agtatccagg 4392ctttgtacag agtgcttttc tgtttagttt ttactttttt
tgttttgttt ttttaaagat 4452gaaataaaga cccaggggga g 4473241255PRTHomo
sapiens 24Met Glu Leu Ala Ala Leu Cys Arg Trp Gly Leu Leu Leu Ala
Leu Leu 1 5 10 15 Pro Pro Gly Ala Ala Ser Thr Gln Val Cys Thr Gly
Thr Asp Met Lys 20 25 30 Leu Arg Leu Pro Ala Ser Pro Glu Thr His
Leu Asp Met Leu Arg His 35 40 45 Leu Tyr Gln Gly Cys Gln Val Val
Gln Gly Asn Leu Glu Leu Thr Tyr 50 55 60 Leu Pro Thr Asn Ala Ser
Leu Ser Phe Leu Gln Asp Ile Gln Glu Val 65 70 75 80 Gln Gly Tyr Val
Leu Ile Ala His Asn Gln Val Arg Gln Val Pro Leu 85 90 95 Gln Arg
Leu Arg Ile Val Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr 100 105 110
Ala Leu Ala Val Leu Asp Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro 115
120 125 Val Thr Gly Ala Ser Pro Gly Gly Leu Arg Glu Leu Gln Leu Arg
Ser 130 135 140 Leu Thr Glu Ile Leu Lys Gly Gly Val Leu Ile Gln Arg
Asn Pro Gln 145 150 155 160 Leu Cys Tyr Gln Asp Thr Ile Leu Trp Lys
Asp Ile Phe His Lys Asn 165 170 175 Asn Gln Leu Ala Leu Thr Leu Ile
Asp Thr Asn Arg Ser Arg Ala Cys 180 185 190 His Pro Cys Ser Pro Met
Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser 195 200 205 Ser Glu Asp Cys
Gln Ser Leu Thr Arg Thr Val Cys Ala Gly Gly Cys 210 215 220 Ala Arg
Cys Lys Gly Pro Leu Pro Thr Asp Cys Cys His Glu Gln Cys 225 230 235
240 Ala Ala Gly Cys Thr Gly Pro Lys His Ser Asp Cys Leu Ala Cys Leu
245 250 255 His Phe Asn His Ser Gly Ile Cys Glu Leu His Cys Pro Ala
Leu Val 260 265 270 Thr Tyr Asn Thr Asp Thr Phe Glu Ser Met Pro Asn
Pro Glu Gly Arg 275 280 285 Tyr Thr Phe Gly Ala Ser Cys Val Thr Ala
Cys Pro Tyr Asn Tyr Leu 290 295 300 Ser Thr Asp Val Gly Ser Cys Thr
Leu Val Cys Pro Leu His Asn Gln 305 310 315 320 Glu Val Thr Ala Glu
Asp Gly Thr Gln Arg Cys Glu Lys Cys Ser Lys 325 330 335 Pro Cys Ala
Arg Val Cys Tyr Gly Leu Gly Met Glu His Leu Arg Glu 340 345 350 Val
Arg Ala Val Thr Ser Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys 355 360
365 Lys Ile Phe Gly Ser Leu Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp
370 375 380 Pro Ala Ser Asn Thr Ala Pro Leu Gln Pro Glu Gln Leu Gln
Val Phe 385 390 395 400 Glu Thr Leu Glu Glu Ile Thr Gly Tyr Leu Tyr
Ile Ser Ala Trp Pro 405 410 415 Asp Ser Leu Pro Asp Leu Ser Val Phe
Gln Asn Leu Gln Val Ile Arg 420 425 430 Gly Arg Ile Leu His Asn Gly
Ala Tyr Ser Leu Thr Leu Gln Gly Leu 435 440 445 Gly Ile Ser Trp Leu
Gly Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly 450 455 460 Leu Ala Leu
Ile His His Asn Thr His Leu Cys Phe Val His Thr Val 465 470 475 480
Pro Trp Asp Gln Leu Phe Arg Asn Pro His Gln Ala Leu Leu His Thr 485
490 495 Ala Asn Arg Pro Glu Asp Glu Cys Val Gly Glu Gly Leu Ala Cys
His 500 505 510 Gln Leu Cys Ala Arg Gly His Cys Trp Gly Pro Gly Pro
Thr Gln Cys 515 520 525 Val Asn Cys Ser Gln Phe Leu Arg Gly Gln Glu
Cys Val Glu Glu Cys 530 535 540 Arg Val Leu Gln Gly Leu Pro Arg Glu
Tyr Val Asn Ala Arg His Cys 545 550 555 560 Leu Pro Cys His Pro Glu
Cys Gln Pro Gln Asn Gly Ser Val Thr Cys 565 570 575 Phe Gly Pro Glu
Ala Asp Gln Cys Val Ala Cys Ala His Tyr Lys Asp 580 585 590 Pro Pro
Phe Cys Val Ala Arg Cys Pro Ser Gly Val Lys Pro Asp Leu 595 600 605
Ser Tyr Met Pro Ile Trp Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln 610
615 620 Pro Cys Pro Ile Asn Cys Thr His Ser Cys Val Asp Leu Asp Asp
Lys 625 630 635 640 Gly Cys Pro Ala Glu Gln Arg Ala Ser Pro Leu Thr
Ser Ile Ile Ser 645 650 655 Ala Val Val Gly Ile Leu Leu Val Val Val
Leu Gly Val Val Phe Gly 660 665 670 Ile Leu Ile Lys Arg Arg Gln Gln
Lys Ile Arg Lys Tyr Thr Met Arg 675 680 685 Arg Leu Leu Gln Glu Thr
Glu Leu Val Glu Pro Leu Thr Pro Ser Gly 690 695 700 Ala Met Pro Asn
Gln Ala Gln Met Arg Ile Leu Lys Glu Thr Glu Leu 705 710 715 720 Arg
Lys Val Lys Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys 725 730
735 Gly Ile Trp Ile Pro Asp Gly Glu Asn Val Lys Ile Pro Val Ala Ile
740 745 750 Lys Val Leu Arg Glu Asn Thr Ser Pro Lys Ala Asn Lys Glu
Ile Leu 755 760 765 Asp Glu Ala Tyr Val Met Ala Gly Val Gly Ser Pro
Tyr Val Ser Arg 770 775 780 Leu Leu Gly Ile Cys Leu Thr Ser Thr Val
Gln Leu Val Thr Gln Leu 785 790 795 800 Met Pro Tyr Gly Cys
Leu Leu Asp His Val Arg Glu Asn Arg Gly Arg 805 810 815 Leu Gly Ser
Gln Asp Leu Leu Asn Trp Cys Met Gln Ile Ala Lys Gly 820 825 830 Met
Ser Tyr Leu Glu Asp Val Arg Leu Val His Arg Asp Leu Ala Ala 835 840
845 Arg Asn Val Leu Val Lys Ser Pro Asn His Val Lys Ile Thr Asp Phe
850 855 860 Gly Leu Ala Arg Leu Leu Asp Ile Asp Glu Thr Glu Tyr His
Ala Asp 865 870 875 880 Gly Gly Lys Val Pro Ile Lys Trp Met Ala Leu
Glu Ser Ile Leu Arg 885 890 895 Arg Arg Phe Thr His Gln Ser Asp Val
Trp Ser Tyr Gly Val Thr Val 900 905 910 Trp Glu Leu Met Thr Phe Gly
Ala Lys Pro Tyr Asp Gly Ile Pro Ala 915 920 925 Arg Glu Ile Pro Asp
Leu Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro 930 935 940 Pro Ile Cys
Thr Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met 945 950 955 960
Ile Asp Ser Glu Cys Arg Pro Arg Phe Arg Glu Leu Val Ser Glu Phe 965
970 975 Ser Arg Met Ala Arg Asp Pro Gln Arg Phe Val Val Ile Gln Asn
Glu 980 985 990 Asp Leu Gly Pro Ala Ser Pro Leu Asp Ser Thr Phe Tyr
Arg Ser Leu 995 1000 1005 Leu Glu Asp Asp Asp Met Gly Asp Leu Val
Asp Ala Glu Glu Tyr 1010 1015 1020 Leu Val Pro Gln Gln Gly Phe Phe
Cys Pro Asp Pro Ala Pro Gly 1025 1030 1035 Ala Gly Gly Met Val His
His Arg His Arg Ser Ser Ser Thr Arg 1040 1045 1050 Ser Gly Gly Gly
Asp Leu Thr Leu Gly Leu Glu Pro Ser Glu Glu 1055 1060 1065 Glu Ala
Pro Arg Ser Pro Leu Ala Pro Ser Glu Gly Ala Gly Ser 1070 1075 1080
Asp Val Phe Asp Gly Asp Leu Gly Met Gly Ala Ala Lys Gly Leu 1085
1090 1095 Gln Ser Leu Pro Thr His Asp Pro Ser Pro Leu Gln Arg Tyr
Ser 1100 1105 1110 Glu Asp Pro Thr Val Pro Leu Pro Ser Glu Thr Asp
Gly Tyr Val 1115 1120 1125 Ala Pro Leu Thr Cys Ser Pro Gln Pro Glu
Tyr Val Asn Gln Pro 1130 1135 1140 Asp Val Arg Pro Gln Pro Pro Ser
Pro Arg Glu Gly Pro Leu Pro 1145 1150 1155 Ala Ala Arg Pro Ala Gly
Ala Thr Leu Glu Arg Pro Lys Thr Leu 1160 1165 1170 Ser Pro Gly Lys
Asn Gly Val Val Lys Asp Val Phe Ala Phe Gly 1175 1180 1185 Gly Ala
Val Glu Asn Pro Glu Tyr Leu Thr Pro Gln Gly Gly Ala 1190 1195 1200
Ala Pro Gln Pro His Pro Pro Pro Ala Phe Ser Pro Ala Phe Asp 1205
1210 1215 Asn Leu Tyr Tyr Trp Asp Gln Asp Pro Pro Glu Arg Gly Ala
Pro 1220 1225 1230 Pro Ser Thr Phe Lys Gly Thr Pro Thr Ala Glu Asn
Pro Glu Tyr 1235 1240 1245 Leu Gly Leu Asp Val Pro Val 1250 1255
251212DNAHomo sapiensCDS(1)..(1212) 25atg aca gcc atc atc aaa gag
atc gtt agc aga aac aaa agg aga tat 48Met Thr Ala Ile Ile Lys Glu
Ile Val Ser Arg Asn Lys Arg Arg Tyr 1 5 10 15 caa gag gat gga ttc
gac tta gac ttg acc tat att tat cca aac att 96Gln Glu Asp Gly Phe
Asp Leu Asp Leu Thr Tyr Ile Tyr Pro Asn Ile 20 25 30 att gct atg
gga ttt cct gca gaa aga ctt gaa ggc gta tac agg aac 144Ile Ala Met
Gly Phe Pro Ala Glu Arg Leu Glu Gly Val Tyr Arg Asn 35 40 45 aat
att gat gat gta gta agg ttt ttg gat tca aag cat aaa aac cat 192Asn
Ile Asp Asp Val Val Arg Phe Leu Asp Ser Lys His Lys Asn His 50 55
60 tac aag ata tac aat ctt tgt gct gaa aga cat tat gac acc gcc aaa
240Tyr Lys Ile Tyr Asn Leu Cys Ala Glu Arg His Tyr Asp Thr Ala Lys
65 70 75 80 ttt aat tgc aga gtt gca caa tat cct ttt gaa gac cat aac
cca cca 288Phe Asn Cys Arg Val Ala Gln Tyr Pro Phe Glu Asp His Asn
Pro Pro 85 90 95 cag cta gaa ctt atc aaa ccc ttt tgt gaa gat ctt
gac caa tgg cta 336Gln Leu Glu Leu Ile Lys Pro Phe Cys Glu Asp Leu
Asp Gln Trp Leu 100 105 110 agt gaa gat gac aat cat gtt gca gca att
cac tgt aaa gct gga aag 384Ser Glu Asp Asp Asn His Val Ala Ala Ile
His Cys Lys Ala Gly Lys 115 120 125 gga cga act ggt gta atg ata tgt
gca tat tta tta cat cgg ggc aaa 432Gly Arg Thr Gly Val Met Ile Cys
Ala Tyr Leu Leu His Arg Gly Lys 130 135 140 ttt tta aag gca caa gag
gcc cta gat ttc tat ggg gaa gta agg acc 480Phe Leu Lys Ala Gln Glu
Ala Leu Asp Phe Tyr Gly Glu Val Arg Thr 145 150 155 160 aga gac aaa
aag gga gta act att ccc agt cag agg cgc tat gtg tat 528Arg Asp Lys
Lys Gly Val Thr Ile Pro Ser Gln Arg Arg Tyr Val Tyr 165 170 175 tat
tat agc tac ctg tta aag aat cat ctg gat tat aga cca gtg gca 576Tyr
Tyr Ser Tyr Leu Leu Lys Asn His Leu Asp Tyr Arg Pro Val Ala 180 185
190 ctg ttg ttt cac aag atg atg ttt gaa act att cca atg ttc agt ggc
624Leu Leu Phe His Lys Met Met Phe Glu Thr Ile Pro Met Phe Ser Gly
195 200 205 gga act tgc aat cct cag ttt gtg gtc tgc cag cta aag gtg
aag ata 672Gly Thr Cys Asn Pro Gln Phe Val Val Cys Gln Leu Lys Val
Lys Ile 210 215 220 tat tcc tcc aat tca gga ccc aca cga cgg gaa gac
aag ttc atg tac 720Tyr Ser Ser Asn Ser Gly Pro Thr Arg Arg Glu Asp
Lys Phe Met Tyr 225 230 235 240 ttt gag ttc cct cag ccg tta cct gtg
tgt ggt gat atc aaa gta gag 768Phe Glu Phe Pro Gln Pro Leu Pro Val
Cys Gly Asp Ile Lys Val Glu 245 250 255 ttc ttc cac aaa cag aac aag
atg cta aaa aag gac aaa atg ttt cac 816Phe Phe His Lys Gln Asn Lys
Met Leu Lys Lys Asp Lys Met Phe His 260 265 270 ttt tgg gta aat aca
ttc ttc ata cca gga cca gag gaa acc tca gaa 864Phe Trp Val Asn Thr
Phe Phe Ile Pro Gly Pro Glu Glu Thr Ser Glu 275 280 285 aaa gta gaa
aat gga agt cta tgt gat caa gaa atc gat agc att tgc 912Lys Val Glu
Asn Gly Ser Leu Cys Asp Gln Glu Ile Asp Ser Ile Cys 290 295 300 agt
ata gag cgt gca gat aat gac aag gaa tat cta gta ctt act tta 960Ser
Ile Glu Arg Ala Asp Asn Asp Lys Glu Tyr Leu Val Leu Thr Leu 305 310
315 320 aca aaa aat gat ctt gac aaa gca aat aaa gac aaa gcc aac cga
tac 1008Thr Lys Asn Asp Leu Asp Lys Ala Asn Lys Asp Lys Ala Asn Arg
Tyr 325 330 335 ttt tct cca aat ttt aag gtg aag ctg tac ttc aca aaa
aca gta gag 1056Phe Ser Pro Asn Phe Lys Val Lys Leu Tyr Phe Thr Lys
Thr Val Glu 340 345 350 gag ccg tca aat cca gag gct agc agt tca act
tct gta aca cca gat 1104Glu Pro Ser Asn Pro Glu Ala Ser Ser Ser Thr
Ser Val Thr Pro Asp 355 360 365 gtt agt gac aat gaa cct gat cat tat
aga tat tct gac acc act gac 1152Val Ser Asp Asn Glu Pro Asp His Tyr
Arg Tyr Ser Asp Thr Thr Asp 370 375 380 tct gat cca gag aat gaa cct
ttt gat gaa gat cag cat aca caa att 1200Ser Asp Pro Glu Asn Glu Pro
Phe Asp Glu Asp Gln His Thr Gln Ile 385 390 395 400 aca aaa gtc tga
1212Thr Lys Val 26403PRTHomo sapiens 26Met Thr Ala Ile Ile Lys Glu
Ile Val Ser Arg Asn Lys Arg Arg Tyr 1 5 10 15 Gln Glu Asp Gly Phe
Asp Leu Asp Leu Thr Tyr Ile Tyr Pro Asn Ile 20 25 30 Ile Ala Met
Gly Phe Pro Ala Glu Arg Leu Glu Gly Val Tyr Arg Asn 35 40 45 Asn
Ile Asp Asp Val Val Arg Phe Leu Asp Ser Lys His Lys Asn His 50 55
60 Tyr Lys Ile Tyr Asn Leu Cys Ala Glu Arg His Tyr Asp Thr Ala Lys
65 70 75 80 Phe Asn Cys Arg Val Ala Gln Tyr Pro Phe Glu Asp His Asn
Pro Pro 85 90 95 Gln Leu Glu Leu Ile Lys Pro Phe Cys Glu Asp Leu
Asp Gln Trp Leu 100 105 110 Ser Glu Asp Asp Asn His Val Ala Ala Ile
His Cys Lys Ala Gly Lys 115 120 125 Gly Arg Thr Gly Val Met Ile Cys
Ala Tyr Leu Leu His Arg Gly Lys 130 135 140 Phe Leu Lys Ala Gln Glu
Ala Leu Asp Phe Tyr Gly Glu Val Arg Thr 145 150 155 160 Arg Asp Lys
Lys Gly Val Thr Ile Pro Ser Gln Arg Arg Tyr Val Tyr 165 170 175 Tyr
Tyr Ser Tyr Leu Leu Lys Asn His Leu Asp Tyr Arg Pro Val Ala 180 185
190 Leu Leu Phe His Lys Met Met Phe Glu Thr Ile Pro Met Phe Ser Gly
195 200 205 Gly Thr Cys Asn Pro Gln Phe Val Val Cys Gln Leu Lys Val
Lys Ile 210 215 220 Tyr Ser Ser Asn Ser Gly Pro Thr Arg Arg Glu Asp
Lys Phe Met Tyr 225 230 235 240 Phe Glu Phe Pro Gln Pro Leu Pro Val
Cys Gly Asp Ile Lys Val Glu 245 250 255 Phe Phe His Lys Gln Asn Lys
Met Leu Lys Lys Asp Lys Met Phe His 260 265 270 Phe Trp Val Asn Thr
Phe Phe Ile Pro Gly Pro Glu Glu Thr Ser Glu 275 280 285 Lys Val Glu
Asn Gly Ser Leu Cys Asp Gln Glu Ile Asp Ser Ile Cys 290 295 300 Ser
Ile Glu Arg Ala Asp Asn Asp Lys Glu Tyr Leu Val Leu Thr Leu 305 310
315 320 Thr Lys Asn Asp Leu Asp Lys Ala Asn Lys Asp Lys Ala Asn Arg
Tyr 325 330 335 Phe Ser Pro Asn Phe Lys Val Lys Leu Tyr Phe Thr Lys
Thr Val Glu 340 345 350 Glu Pro Ser Asn Pro Glu Ala Ser Ser Ser Thr
Ser Val Thr Pro Asp 355 360 365 Val Ser Asp Asn Glu Pro Asp His Tyr
Arg Tyr Ser Asp Thr Thr Asp 370 375 380 Ser Asp Pro Glu Asn Glu Pro
Phe Asp Glu Asp Gln His Thr Gln Ile 385 390 395 400 Thr Lys Val
27597DNAHomo sapiensCDS(1)..(597) 27atg tca aac gtg cga gtg tct aac
ggg agc cct agc ctg gag cgg atg 48Met Ser Asn Val Arg Val Ser Asn
Gly Ser Pro Ser Leu Glu Arg Met 1 5 10 15 gac gcc agg cag gcg gag
cac ccc aag ccc tcg gcc tgc agg aac ctc 96Asp Ala Arg Gln Ala Glu
His Pro Lys Pro Ser Ala Cys Arg Asn Leu 20 25 30 ttc ggc ccg gtg
gac cac gaa gag tta acc cgg gac ttg gag aag cac 144Phe Gly Pro Val
Asp His Glu Glu Leu Thr Arg Asp Leu Glu Lys His 35 40 45 tgc aga
gac atg gaa gag gcg agc cag cgc aag tgg aat ttc gat ttt 192Cys Arg
Asp Met Glu Glu Ala Ser Gln Arg Lys Trp Asn Phe Asp Phe 50 55 60
cag aat cac aaa ccc cta gag ggc aag tac gag tgg caa gag gtg gag
240Gln Asn His Lys Pro Leu Glu Gly Lys Tyr Glu Trp Gln Glu Val Glu
65 70 75 80 aag ggc agc ttg ccc gag ttc tac tac aga ccc ccg cgg ccc
ccc aaa 288Lys Gly Ser Leu Pro Glu Phe Tyr Tyr Arg Pro Pro Arg Pro
Pro Lys 85 90 95 ggt gcc tgc aag gtg ccg gcg cag gag agc cag gat
gtc agc ggg agc 336Gly Ala Cys Lys Val Pro Ala Gln Glu Ser Gln Asp
Val Ser Gly Ser 100 105 110 cgc ccg gcg gcg cct tta att ggg gct ccg
gct aac tct gag gac acg 384Arg Pro Ala Ala Pro Leu Ile Gly Ala Pro
Ala Asn Ser Glu Asp Thr 115 120 125 cat ttg gtg gac cca aag act gat
ccg tcg gac agc cag acg ggg tta 432His Leu Val Asp Pro Lys Thr Asp
Pro Ser Asp Ser Gln Thr Gly Leu 130 135 140 gcg gag caa tgc gca gga
ata agg aag cga cct gca acc gac gat tct 480Ala Glu Gln Cys Ala Gly
Ile Arg Lys Arg Pro Ala Thr Asp Asp Ser 145 150 155 160 tct act caa
aac aaa aga gcc aac aga aca gaa gaa aat gtt tca gac 528Ser Thr Gln
Asn Lys Arg Ala Asn Arg Thr Glu Glu Asn Val Ser Asp 165 170 175 ggt
tcc cca aat gcc ggt tct gtg gag cag acg ccc aag aag cct ggc 576Gly
Ser Pro Asn Ala Gly Ser Val Glu Gln Thr Pro Lys Lys Pro Gly 180 185
190 ctc aga aga cgt caa acg taa 597Leu Arg Arg Arg Gln Thr 195
28198PRTHomo sapiens 28Met Ser Asn Val Arg Val Ser Asn Gly Ser Pro
Ser Leu Glu Arg Met 1 5 10 15 Asp Ala Arg Gln Ala Glu His Pro Lys
Pro Ser Ala Cys Arg Asn Leu 20 25 30 Phe Gly Pro Val Asp His Glu
Glu Leu Thr Arg Asp Leu Glu Lys His 35 40 45 Cys Arg Asp Met Glu
Glu Ala Ser Gln Arg Lys Trp Asn Phe Asp Phe 50 55 60 Gln Asn His
Lys Pro Leu Glu Gly Lys Tyr Glu Trp Gln Glu Val Glu 65 70 75 80 Lys
Gly Ser Leu Pro Glu Phe Tyr Tyr Arg Pro Pro Arg Pro Pro Lys 85 90
95 Gly Ala Cys Lys Val Pro Ala Gln Glu Ser Gln Asp Val Ser Gly Ser
100 105 110 Arg Pro Ala Ala Pro Leu Ile Gly Ala Pro Ala Asn Ser Glu
Asp Thr 115 120 125 His Leu Val Asp Pro Lys Thr Asp Pro Ser Asp Ser
Gln Thr Gly Leu 130 135 140 Ala Glu Gln Cys Ala Gly Ile Arg Lys Arg
Pro Ala Thr Asp Asp Ser 145 150 155 160 Ser Thr Gln Asn Lys Arg Ala
Asn Arg Thr Glu Glu Asn Val Ser Asp 165 170 175 Gly Ser Pro Asn Ala
Gly Ser Val Glu Gln Thr Pro Lys Lys Pro Gly 180 185 190 Leu Arg Arg
Arg Gln Thr 195 29894DNAHomo sapiensCDS(1)..(894) 29atg aca aca ccc
aga aat tca gta aat ggg act ttc ccg gca gag cca 48Met Thr Thr Pro
Arg Asn Ser Val Asn Gly Thr Phe Pro Ala Glu Pro 1 5 10 15 atg aaa
ggc cct att gct atg caa tct ggt cca aaa cca ctc ttc agg 96Met Lys
Gly Pro Ile Ala Met Gln Ser Gly Pro Lys Pro Leu Phe Arg 20 25 30
agg atg tct tca ctg gtg ggc ccc acg caa agc ttc ttc atg agg gaa
144Arg Met Ser Ser Leu Val Gly Pro Thr Gln Ser Phe Phe Met Arg Glu
35 40 45 tct aag act ttg ggg gct gtc cag att atg aat ggg ctc ttc
cac att 192Ser Lys Thr Leu Gly Ala Val Gln Ile Met Asn Gly Leu Phe
His Ile 50 55 60 gcc ctg ggg ggt ctt ctg atg atc cca gca ggg atc
tat gca ccc atc 240Ala Leu Gly Gly Leu Leu Met Ile Pro Ala Gly Ile
Tyr Ala Pro Ile 65 70 75 80 tgt gtg act gtg tgg tac cct ctc tgg gga
ggc att atg tat att att 288Cys Val Thr Val Trp Tyr Pro Leu Trp Gly
Gly Ile Met Tyr Ile Ile
85 90 95 tcc gga tca ctc ttg gca gca acg gag aaa aac tct agg aag
tgt ttg 336Ser Gly Ser Leu Leu Ala Ala Thr Glu Lys Asn Ser Arg Lys
Cys Leu 100 105 110 gtc aaa gga aaa atg ata atg aat tca ttg agc ctc
ttt gct gcc att 384Val Lys Gly Lys Met Ile Met Asn Ser Leu Ser Leu
Phe Ala Ala Ile 115 120 125 tct gga atg att ctt tca atc atg gac ata
ctt aat att aaa att tcc 432Ser Gly Met Ile Leu Ser Ile Met Asp Ile
Leu Asn Ile Lys Ile Ser 130 135 140 cat ttt tta aaa atg gag agt ctg
aat ttt att aga gct cac aca cca 480His Phe Leu Lys Met Glu Ser Leu
Asn Phe Ile Arg Ala His Thr Pro 145 150 155 160 tat att aac ata tac
aac tgt gaa cca gct aat ccc tct gag aaa aac 528Tyr Ile Asn Ile Tyr
Asn Cys Glu Pro Ala Asn Pro Ser Glu Lys Asn 165 170 175 tcc cca tct
acc caa tac tgt tac agc ata caa tct ctg ttc ttg ggc 576Ser Pro Ser
Thr Gln Tyr Cys Tyr Ser Ile Gln Ser Leu Phe Leu Gly 180 185 190 att
ttg tca gtg atg ctg atc ttt gcc ttc ttc cag gaa ctt gta ata 624Ile
Leu Ser Val Met Leu Ile Phe Ala Phe Phe Gln Glu Leu Val Ile 195 200
205 gct ggc atc gtt gag aat gaa tgg aaa aga acg tgc tcc aga ccc aaa
672Ala Gly Ile Val Glu Asn Glu Trp Lys Arg Thr Cys Ser Arg Pro Lys
210 215 220 tct aac ata gtt ctc ctg tca gca gaa gaa aaa aaa gaa cag
act att 720Ser Asn Ile Val Leu Leu Ser Ala Glu Glu Lys Lys Glu Gln
Thr Ile 225 230 235 240 gaa ata aaa gaa gaa gtg gtt ggg cta act gaa
aca tct tcc caa cca 768Glu Ile Lys Glu Glu Val Val Gly Leu Thr Glu
Thr Ser Ser Gln Pro 245 250 255 aag aat gaa gaa gac att gaa att att
cca atc caa gaa gag gaa gaa 816Lys Asn Glu Glu Asp Ile Glu Ile Ile
Pro Ile Gln Glu Glu Glu Glu 260 265 270 gaa gaa aca gag acg aac ttt
cca gaa cct ccc caa gat cag gaa tcc 864Glu Glu Thr Glu Thr Asn Phe
Pro Glu Pro Pro Gln Asp Gln Glu Ser 275 280 285 tca cca ata gaa aat
gac agc tct cct taa 894Ser Pro Ile Glu Asn Asp Ser Ser Pro 290 295
30297PRTHomo sapiens 30Met Thr Thr Pro Arg Asn Ser Val Asn Gly Thr
Phe Pro Ala Glu Pro 1 5 10 15 Met Lys Gly Pro Ile Ala Met Gln Ser
Gly Pro Lys Pro Leu Phe Arg 20 25 30 Arg Met Ser Ser Leu Val Gly
Pro Thr Gln Ser Phe Phe Met Arg Glu 35 40 45 Ser Lys Thr Leu Gly
Ala Val Gln Ile Met Asn Gly Leu Phe His Ile 50 55 60 Ala Leu Gly
Gly Leu Leu Met Ile Pro Ala Gly Ile Tyr Ala Pro Ile 65 70 75 80 Cys
Val Thr Val Trp Tyr Pro Leu Trp Gly Gly Ile Met Tyr Ile Ile 85 90
95 Ser Gly Ser Leu Leu Ala Ala Thr Glu Lys Asn Ser Arg Lys Cys Leu
100 105 110 Val Lys Gly Lys Met Ile Met Asn Ser Leu Ser Leu Phe Ala
Ala Ile 115 120 125 Ser Gly Met Ile Leu Ser Ile Met Asp Ile Leu Asn
Ile Lys Ile Ser 130 135 140 His Phe Leu Lys Met Glu Ser Leu Asn Phe
Ile Arg Ala His Thr Pro 145 150 155 160 Tyr Ile Asn Ile Tyr Asn Cys
Glu Pro Ala Asn Pro Ser Glu Lys Asn 165 170 175 Ser Pro Ser Thr Gln
Tyr Cys Tyr Ser Ile Gln Ser Leu Phe Leu Gly 180 185 190 Ile Leu Ser
Val Met Leu Ile Phe Ala Phe Phe Gln Glu Leu Val Ile 195 200 205 Ala
Gly Ile Val Glu Asn Glu Trp Lys Arg Thr Cys Ser Arg Pro Lys 210 215
220 Ser Asn Ile Val Leu Leu Ser Ala Glu Glu Lys Lys Glu Gln Thr Ile
225 230 235 240 Glu Ile Lys Glu Glu Val Val Gly Leu Thr Glu Thr Ser
Ser Gln Pro 245 250 255 Lys Asn Glu Glu Asp Ile Glu Ile Ile Pro Ile
Gln Glu Glu Glu Glu 260 265 270 Glu Glu Thr Glu Thr Asn Phe Pro Glu
Pro Pro Gln Asp Gln Glu Ser 275 280 285 Ser Pro Ile Glu Asn Asp Ser
Ser Pro 290 295 31596DNAhomo sapiens 31acgcgtactg gagtcaatga
aagcaactat ttcaaaagat cagattactt accagtttca 60ctaataaaga tttattactt
taaaccttta tcataaaatg tatgctttga atactgtgaa 120gtacactgca
tataaggagt gtggtatagt ataaagaaac tttctgcagg tagtaattat
180agtgaagatt ttaggtttac aaagccctag ctgttttctg tgtagctttt
attattctta 240tgactcttga caagtttgta gcttcaccat atacatttaa
tattttgcaa taattggcct 300tgttcctgag ctgttggatt cggggccgta
gcactgtctg agaggtttac atttctcaca 360gtgaaccggt ctctttttca
gctgcttcct ggcttctttt tactcaggtt tccactgctt 420ttttgctttt
tttaatgctg tatgaaggtg ttaacatttg tttatatttt tcattaattg
480taataccttt aaatcatgca tcatactcag aaatagggat tagaatttaa
gtgacatctt 540tggcctaata taatttacct gttaaaaatt tgtgaaagct
attgcttagc ggccgc 59632511DNAHomo sapiens 32acgcgtccat gtccgtacct
ttctagttca taccttcttt taattttttt tttcttttca 60atttgaagag agtgcttcct
ctgttcttaa ggctagggaa ccaaattagg ttgtttcaat 120atcgtgctaa
aagatactgc ctttagaaga aggctattga caatccagcg tgtctcggtg
180gaactctgac tccatggttc actttcatga tggccacatg cctcctgccc
agagcccggc 240agccactgtg cagtgggaag gggggccgat acactgtacg
agagtgagta gcaggtctca 300cagtgaaccg gtctctttcc ctactgtgtc
acactcctaa tggaatgccg ttatccaaag 360agcagcacga acccgacagg
gctgagtggc ttgtgctagg gagaggtttg tgtcattcct 420gctgaccaaa
ctgcaggaaa aactgctaat tgtcatgctg aagactgcct gacggggaga
480ctctgccttc tgtaagtagg tcagcggccg c 51133203DNAHomo sapiens
33acgcgtaatt catatttgca tgtcgctatg tgttctggga aatcaccata aacgtgaaat
60gtctttggat ttgggaatct tataagttct gtatgagacc actcggatga gctgttggat
120tcggggccgt agcactgtct gagaggttta catttctcac agtgaaccgg
tctctttttc 180agctgcttct tttttgcggc cgc 20334205DNAHomo sapiens
34gcggccgcaa ttcatatttg catgtcgcta tgtgttctgg gaaatcacca taaacgtgaa
60atgtctttgg atttgggaat cttataagtt ctgtatgaga ccactcggat gagctgttgg
120attcggggcc gtagcactgt ctgagaggtt tacatttctc acagtgaacc
ggtctctttt 180tcagctgctt cttttttgcg gccgc 2053545DNAArtificial
SequenceDNA target sequence of the miR-142-3p 35gcggccgcgt
cgactccata aagtaggaaa cactacagcg gccgc 4536128DNAArtificial
SequenceDNA target sequence four time repeat miR-142-3pT4X
36gcggccgcgt cgactccata aagtaggaaa cactacacga ttccataaag taggaaacac
60tacaaccggt tccataaagt aggaaacact acatcactcc ataaagtagg aaacactaca
120gcggccgc 128371131DNAherpes simplex virus 1 37atggcttcgt
accccggcca tcagcacgcg tctgcgttcg accaggctgc gcgttctcgc 60ggccatagca
accgacgtac ggcgttgcgc cctcgccggc agcaagaagc cacggaagtc
120cgcccggagc agaaaatgcc cacgctactg cgggtttata tagacggtcc
ccacgggatg 180gggaaaacca ccaccacgca actgctggtg gccctgggtt
cgcgcgacga tatcgtctac 240gtacccgagc cgatgactta ctggcaggtg
ctgggggctt ccgagacaat cgcgaacatc 300tacaccacac aacaccgcct
cgaccagggt gagatatcgg ccggggacgc ggcggtggta 360atgacaagcg
cccagataac aatgggcatg ccttatgccg tgaccgacgc cgttctggct
420cctcatatcg ggggggaggc tgggagctca catgccccgc ccccggccct
caccctcatc 480ttcgaccgcc atcccatcgc cgccctcctg tgttacccgg
ccgcgcgata ccttatgggc 540agcatgaccc cccaggccgt gctggcgttc
gtggccctca tcccgccgac cttgcccggc 600acaaacatcg tgttgggggc
ccttccggag gacagacaca tcgaccgcct ggccaaacgc 660cagcgccccg
gcgagcggct tgacctggct atgctggccg cgattcgccg cgtttacgag
720ctgcttgcca atacggtgcg gtatctgcag ggcggcgggt cgtggcggga
ggattgggga 780cagctttcgg ggacggccgt gccgccccag ggtgccgagc
cccagagcaa cgcgggccca 840cgaccccata tcggggacac gttatttacc
ctgtttcggg cccccgagtt gctggccccc 900aacggcgacc tgtataacgt
gtttgcctgg gccttggacg tcttggccaa acgcctccgt 960cccatgcacg
tctttatcct ggattacgac caatcgcccg ccggctaccg ggacgccctg
1020ctgcaactta cctccgggat ggtccagacc cacgtcacca cccccggctc
cataccgacg 1080atctgcgacc tggcgcgcac gtttgcccgg gagatggggg
aggctaacta a 113138499DNAHomo sapiens 38atgaaatata caagttatat
cttggctttt cagctctgca tcgttttggg ttctcttggc 60tgttactgcc aggaccatat
gtaaaagaag cagaaaacct taagaaatat tttaatgcag 120gtcattcaga
tgtagcggat aatggaactc ttttcttagg cattttgaag aattggaaag
180aggagagtga cagaaaaata atgcagagcc aaattgtctc cttttacttc
aaacttttta 240aaaactttaa agatgaccag agcatccaaa agagtgtgga
gaccatcaag gaagacatga 300atgtaagttt ttcaatagca acaaaaagaa
acgagatgac ttcgaaaagc tgactaatta 360ttcggtaact gacttgaatg
tccaacgcaa agcaatacat gaactcatcc aagtgatggc 420tgaactgtcg
ccagcagcta aaacagggaa gcgaaaaagg agtcagatgc tgtttcgagg
480tcgaagagca tcccagtaa 49939468DNAMus musculus 39atgaacgcta
cacactgcat cttggctttg cagctcttcc tcatggctgt ttctggctgt 60tactgccacg
gcacagtcat tgaaagccta gaaagtctga ataactattt taactcaagt
120ggcatagatg tggaagaaaa gagtctcttc ttggatatct ggaggaactg
gcaaaaggat 180ggtgacatga aaatcctgca gagccagatt atctctttct
acctcagact ctttgaagtc 240ttgaaagaca atcaggccat cagcaacaac
ataagcgtca ttgaatcaca cctgattact 300accttcttca gcaacagcaa
ggcgaaaaag gatgcattca tgagtattgc caagtttgag 360gtcaacaacc
cacaggtcca gcgccaagca ttcaatgagc tcatccgagt ggtccaccag
420ctgttgccgg aatccagcct caggaagcgg aaaaggagtc gctgctga
46840462DNAHomo sapiens 40atgtacagga tgcaactcct gtcttgcatt
gcactaagtc ttgcacttgt cacaaacagt 60gcacctactt caagttctac aaagaaaaca
cagctacaac tggagcattt actgctggat 120ttacagatga ttttgaatgg
aattaataat tacaagaatc ccaaactcac caggatgctc 180acatttaagt
tttacatgcc caagaaggcc acagaactga aacatcttca gtgtctagaa
240gaagaactca aacctctgga ggaagtgcta aatttagctc aaagcaaaaa
ctttcactta 300agacccaggg acttaatcag caatatcaac gtaatagttc
tggaactaaa gggatctgaa 360acaacattca tgtgtgaata tgctgatgag
acagcaacca ttgtagaatt tctgaacaga 420tggattacct tttgtcaaag
catcatctca acactgactt ga 4624123RNAArtificial
SequenceOligonucleotide primer 41uguaguguuu ccuacuuuau gga
234223RNAArtificial SequenceOligonucleotide primer 42uguaguguuu
ccuacuuuau gga 2343546DNAArtificial SequenceYeast cytosine
deaminase with cloning sites 43aacacgatta taaatggtga cagggggaat
ggcaagcaag tgggatcaga agggtatgga 60cattgcctat gaggaggcgg ccttaggtta
caaagagggt ggtgttccta ttggcggatg 120tcttatcaat aacaaagacg
gaagtgttct cggtcgtggt cacaacatga gatttcaaaa 180gggatccgcc
acactacatg gtgagatctc cactttggaa aactgtggga gattagaggg
240caaagtgtac aaagatacca ctttgtatac gacgctgtct ccatgcgaca
tgtgtacagg 300tgccatcatc atgtatggta ttccacgctg tgttgtcggt
gagaacgtta atttcaaaag 360taagggcgag aaatatttac aaactagagg
tcacgaggtt gttgttgttg acgatgagag 420gtgtaaaaag atcatgaaac
aatttatcga tgaaagacct caggattggt ttgaagatat 480tggtgagtag
gcggccgcgc catagataaa ataaaagatt ttatttagtc tccagaaaaa 540gggggg
54644537DNAArtificial SequenceCD codon optimized with cloning sites
44ttataaatgg tgaccggcgg catggcctcc aagtgggatc aaaagggcat ggatatcgct
60tacgaggagg ccgccctggg ctacaaggag ggcggcgtgc ctatcggcgg ctgtctgatc
120aacaacaagg acggcagtgt gctgggcagg ggccacaaca tgaggttcca
gaagggctcc 180gccaccctgc acggcgagat ctccaccctg gagaactgtg
gcaggctgga gggcaaggtg 240tacaaggaca ccaccctgta caccaccctg
tccccttgtg acatgtgtac cggcgctatc 300atcatgtacg gcatccctag
gtgtgtggtg ggcgagaacg tgaacttcaa gtccaagggc 360gagaagtacc
tgcaaaccag gggccacgag gtggtggttg ttgacgatga gaggtgtaag
420aagatcatga agcagttcat cgacgagagg cctcaggact ggttcgagga
tatcggcgag 480tgataagcgg ccgcagataa aataaaagat tttatttagt
ctccagaaaa agggggg 5374540DNAArtificial SequenceOligonucleotide
primer 45tcgaggatat cggcgagtga aacccgttat tctttttggc
404640DNAArtificial SequenceOligonucleotide Primer 46gccaaaaaga
ataacgggtt tcactcgccg atatcctcga 404782DNAArtificial
SequenceOligonucleotide primer 47tcggcgagtg atccggcggc ggcgcctccg
gcggcggcgc ctccggcggc ggcgcctccg 60gcggcggcgc caacccgtta tt
824882DNAArtificial SequenceOligonucleotide Primer 48aataacgggt
tggcgccgcc gccggaggcg ccgccgccgg aggcgccgcc gccggaggcg 60ccgccgccgg
atcactcgcc ga 8249480DNAArtificial SequenceCytosine deaminase codon
optimized heat stabilized 49atggtgaccg gcggcatggc ctccaagtgg
gatcaaaagg gcatggatat cgcttacgag 60gaggccctgc tgggctacaa ggagggcggc
gtgcctatcg gcggctgtct gatcaacaac 120aaggacggca gtgtgctggg
caggggccac aacatgaggt tccagaaggg ctccgccacc 180ctgcacggcg
agatctccac cctggagaac tgtggcaggc tggagggcaa ggtgtacaag
240gacaccaccc tgtacaccac cctgtcccct tgtgacatgt gtaccggcgc
tatcatcatg 300tacggcatcc ctaggtgtgt gatcggcgag aacgtgaact
tcaagtccaa gggcgagaag 360tacctgcaaa ccaggggcca cgaggtggtg
gttgttgacg atgagaggtg taagaagctg 420atgaagcagt tcatcgacga
gaggcctcag gactggttcg aggatatcgg cgagtgataa 48050546DNAArtificial
SequenceCytosine deaminase codon optimized heat stabilized
50aacacgatta taaatggtga ccggcggcat ggcctccaag tgggatcaaa agggcatgga
60tatcgcttac gaggaggccc tgctgggcta caaggagggc ggcgtgccta tcggcggctg
120tctgatcaac aacaaggacg gcagtgtgct gggcaggggc cacaacatga
ggttccagaa 180gggctccgcc accctgcacg gcgagatctc caccctggag
aactgtggca ggctggaggg 240caaggtgtac aaggacacca ccctgtacac
caccctgtcc ccttgtgaca tgtgtaccgg 300cgctatcatc atgtacggca
tccctaggtg tgtgatcggc gagaacgtga acttcaagtc 360caagggcgag
aagtacctgc aaaccagggg ccacgaggtg gtggttgttg acgatgagag
420gtgtaagaag ctgatgaagc agttcatcga cgagaggcct caggactggt
tcgaggatat 480cggcgagtaa gcggccgcgc catagataaa ataaaagatt
ttatttagtc tccagaaaaa 540gggggg 5465140DNAArtificial
SequenceOligonucleotide primer 51tcgaggatat cggcgagtga aacccgttat
tctttttggc 405240DNAArtificial SequenceOligonucleotide Primer
52gccaaaaaga ataacgggtt tcactcgccg atatcctcga 405352DNAArtificial
SequenceOligonucleotide primer complement 53ccaagctcct atagccgctc
actatctact tgggcaataa gaaaaaccga ag 525452DNAArtificial
SequenceOligonucleotide primer complement 54ggttcgagga tatcggcgag
tgatagatga acccgttatt ctttttggct tc 52551296DNAArtificial
SequenceCD-UPRT cloning sequence 55aacacgatta taaatggtga ccggcggcat
ggcctccaag tgggatcaaa agggcatgga 60tatcgcttac gaggaggccc tgctgggcta
caaggagggc ggcgtgccta tcggcggctg 120tctgatcaac aacaaggacg
gcagtgtgct gggcaggggc cacaacatga ggttccagaa 180gggctccgcc
accctgcacg gcgagatctc caccctggag aactgtggca ggctggaggg
240caaggtgtac aaggacacca ccctgtacac caccctgtcc ccttgtgaca
tgtgtaccgg 300cgctatcatc atgtacggca tccctaggtg tgtgatcggc
gagaacgtga acttcaagtc 360caagggcgag aagtacctgc aaaccagggg
ccacgaggtg gtggttgttg acgatgagag 420gtgtaagaag ctgatgaagc
agttcatcga cgagaggcct caggactggt tcgaggatat 480cggcgagaac
ccgttattct ttttggcttc tccattcttg taccttacat atcttatata
540ttatccaaac aaagggtctt tcgttagcaa acctagaaat ctgcaaaaaa
tgtcttcgga 600accatttaag aacgtctact tgctacctca aacaaaccaa
ttgctgggtt tgtacaccat 660catcagaaat aagaatacaa ctagacctga
tttcattttc tactccgata gaatcatcag 720attgttggtt gaagaaggtt
tgaaccatct acctgtgcaa aagcaaattg tggaaactga 780caccaacgaa
aacttcgaag gtgtctcatt catgggtaaa atctgtggtg tttccattgt
840cagagctggt gaatcgatgg agcaaggatt aagagactgt tgtaggtctg
tgcgtatcgg 900taaaatttta attcaaaggg acgaggagac tgctttacca
aagttattct acgaaaaatt 960accagaggat atatctgaaa ggtatgtctt
cctattagac ccaatgctgg ccaccggtgg 1020tagtgctatc atggctacag
aagtcttgat taagagaggt gttaagccag agagaattta 1080cttcttaaac
ctaatctgta gtaaggaagg gattgaaaaa taccatgccg ccttcccaga
1140ggtcagaatt gttactggtg ccctcgacag aggtctagat gaaaacaagt
atctagttcc 1200agggttgggt gactttggtg acagatacta ctgtgtttaa
gcggccgcgc catagataaa 1260ataaaagatt ttatttagtc tccagaaaaa gggggg
1296565PRTArtificial SequencePeptide linker sequence 56Ser Gly Gly
Gly Gly 1 5 5782DNAArtificial SequenceOligonucleotide primer
57tcggcgagtg atccggcggc ggcgcctccg gcggcggcgc ctccggcggc ggcgcctccg
60gcggcggcgc caacccgtta tt 825882DNAArtificial
SequenceOligonucleotide primer 58aataacgggt tggcgccgcc gccggaggcg
ccgccgccgg aggcgccgcc gccggaggcg 60ccgccgccgg atcactcgcc ga
82591356DNAArtificial SequenceCD codon optimized heat stabilized
linker UPRT 59aacacgatta taaatggtga ccggcggcat ggcctccaag
tgggatcaaa agggcatgga 60tatcgcttac gaggaggccc tgctgggcta caaggagggc
ggcgtgccta tcggcggctg 120tctgatcaac aacaaggacg gcagtgtgct
gggcaggggc cacaacatga ggttccagaa 180gggctccgcc accctgcacg
gcgagatctc caccctggag aactgtggca ggctggaggg 240caaggtgtac
aaggacacca ccctgtacac
caccctgtcc ccttgtgaca tgtgtaccgg 300cgctatcatc atgtacggca
tccctaggtg tgtgatcggc gagaacgtga acttcaagtc 360caagggcgag
aagtacctgc aaaccagggg ccacgaggtg gtggttgttg acgatgagag
420gtgtaagaag ctgatgaagc agttcatcga cgagaggcct caggactggt
tcgaggatat 480cggcgagtcc ggcggcggcg cctccggcgg cggcgcctcc
ggcggcggcg cctccggcgg 540cggcgccaac ccgttattct ttttggcttc
tccattcttg taccttacat atcttatata 600ttatccaaac aaagggtctt
tcgttagcaa acctagaaat ctgcaaaaaa tgtcttcgga 660accatttaag
aacgtctact tgctacctca aacaaaccaa ttgctgggtt tgtacaccat
720catcagaaat aagaatacaa ctagacctga tttcattttc tactccgata
gaatcatcag 780attgttggtt gaagaaggtt tgaaccatct acctgtgcaa
aagcaaattg tggaaactga 840caccaacgaa aacttcgaag gtgtctcatt
catgggtaaa atctgtggtg tttccattgt 900cagagctggt gaatcgatgg
agcaaggatt aagagactgt tgtaggtctg tgcgtatcgg 960taaaatttta
attcaaaggg acgaggagac tgctttacca aagttattct acgaaaaatt
1020accagaggat atatctgaaa ggtatgtctt cctattagac ccaatgctgg
ccaccggtgg 1080tagtgctatc atggctacag aagtcttgat taagagaggt
gttaagccag agagaattta 1140cttcttaaac ctaatctgta gtaaggaagg
gattgaaaaa taccatgccg ccttcccaga 1200ggtcagaatt gttactggtg
ccctcgacag aggtctagat gaaaacaagt atctagttcc 1260agggttgggt
gactttggtg acagatacta ctgtgtttaa gcggccgcgc catagataaa
1320ataaaagatt ttatttagtc tccagaaaaa gggggg 1356601269DNAArtificial
SequenceCD codon optimized heat stabilized OPRT 60aacacgatta
taaatggtga ccggcggcat ggcctccaag tgggatcaaa agggcatgga 60tatcgcttac
gaggaggccc tgctgggcta caaggagggc ggcgtgccta tcggcggctg
120tctgatcaac aacaaggacg gcagtgtgct gggcaggggc cacaacatga
ggttccagaa 180gggctccgcc accctgcacg gcgagatctc caccctggag
aactgtggca ggctggaggg 240caaggtgtac aaggacacca ccctgtacac
caccctgtcc ccttgtgaca tgtgtaccgg 300cgctatcatc atgtacggca
tccctaggtg tgtgatcggc gagaacgtga acttcaagtc 360caagggcgag
aagtacctgc aaaccagggg ccacgaggtg gtggttgttg acgatgagag
420gtgtaagaag ctgatgaagc agttcatcga cgagaggcct caggactggt
tcgaggatat 480cggcgaggcg gtcgctcgtg cagctttggg gccattggtg
acgggtctgt acgacgtgca 540ggctttcaag tttggggact tcgtgctgaa
gagcgggctt tcctccccca tctacatcga 600tctgcggggc atcgtgtctc
gaccgcgtct tctgagtcag gttgcagata ttttattcca 660aactgcccaa
aatgcaggca tcagttttga caccgtgtgt ggagtgcctt atacagcttt
720gccattggct acagttatct gttcaaccaa tcaaattcca atgcttatta
gaaggaaaga 780aacaaaggat tatggaacta agcgtcttgt agaaggaact
attaatccag gagaaacctg 840tttaatcatt gaagatgttg tcaccagtgg
atctagtgtt ttggaaactg ttgaggttct 900tcagaaggag ggcttgaagg
tcactgatgc catagtgctg ttggacagag agcagggagg 960caaggacaag
ttgcaggcgc acgggatccg cctccactca gtgtgtacat tgtccaaaat
1020gctggagatt ctcgagcagc agaaaaaagt tgatgctgag acagttggga
gagtgaagag 1080gtttattcag gagaatgtct ttgtggcagc gaatcataat
ggttctcccc tttctataaa 1140ggaagcaccc aaagaactca gcttcggtgc
acgtgcagag ctgcccagga tccacccagt 1200tgcatcgaag taagcggccg
cgccatagat aaaataaaag attttattta gtctccagaa 1260aaagggggg
1269611329DNAArtificial SequenceCD codon optimized heat stabilized
linker OPRT 61aacacgatta taaatggtga ccggcggcat ggcctccaag
tgggatcaaa agggcatgga 60tatcgcttac gaggaggccc tgctgggcta caaggagggc
ggcgtgccta tcggcggctg 120tctgatcaac aacaaggacg gcagtgtgct
gggcaggggc cacaacatga ggttccagaa 180gggctccgcc accctgcacg
gcgagatctc caccctggag aactgtggca ggctggaggg 240caaggtgtac
aaggacacca ccctgtacac caccctgtcc ccttgtgaca tgtgtaccgg
300cgctatcatc atgtacggca tccctaggtg tgtgatcggc gagaacgtga
acttcaagtc 360caagggcgag aagtacctgc aaaccagggg ccacgaggtg
gtggttgttg acgatgagag 420gtgtaagaag ctgatgaagc agttcatcga
cgagaggcct caggactggt tcgaggatat 480cggcgagtcc ggcggcggcg
cctccggcgg cggcgcctcc ggcggcggcg cctccggcgg 540cggcgccgcg
gtcgctcgtg cagctttggg gccattggtg acgggtctgt acgacgtgca
600ggctttcaag tttggggact tcgtgctgaa gagcgggctt tcctccccca
tctacatcga 660tctgcggggc atcgtgtctc gaccgcgtct tctgagtcag
gttgcagata ttttattcca 720aactgcccaa aatgcaggca tcagttttga
caccgtgtgt ggagtgcctt atacagcttt 780gccattggct acagttatct
gttcaaccaa tcaaattcca atgcttatta gaaggaaaga 840aacaaaggat
tatggaacta agcgtcttgt agaaggaact attaatccag gagaaacctg
900tttaatcatt gaagatgttg tcaccagtgg atctagtgtt ttggaaactg
ttgaggttct 960tcagaaggag ggcttgaagg tcactgatgc catagtgctg
ttggacagag agcagggagg 1020caaggacaag ttgcaggcgc acgggatccg
cctccactca gtgtgtacat tgtccaaaat 1080gctggagatt ctcgagcagc
agaaaaaagt tgatgctgag acagttggga gagtgaagag 1140gtttattcag
gagaatgtct ttgtggcagc gaatcataat ggttctcccc tttctataaa
1200ggaagcaccc aaagaactca gcttcggtgc acgtgcagag ctgcccagga
tccacccagt 1260tgcatcgaag taagcggccg cgccatagat aaaataaaag
attttattta gtctccagaa 1320aaagggggg 13296223DNAArtificial
SequenceOligonucleotide primer 62ctgatcttac tctttggacc ttg
236324DNAArtificial SequenceOligonucleotide primer 63cccctttttc
tggagactaa ataa 246419DNAArtificial SequenceOligonucleotide primer
64agcccacaac ccctcactc 196518DNAArtificial SequenceOligonucleotide
primer 65tctcccgatc ccggacga 186626DNAArtificial
SequenceOligonucleotide primer 66ccccaaatga aagacccccg ctgacg
266723DNAArtificial SequenceOligonucleotide primer 67atcatcatgt
acggcatccc tag 236824DNAArtificial SequenceOligonucleotide primer
68tgaactgctt catcagcttc ttac 246925DNAArtificial
SequenceOligonucleotide primer 69tcatcgtcaa caaccaccac ctcgt
257022DNAArtificial SequenceOligonucleotide primer 70aacctcaacc
tcccctacaa gt 227120DNAArtificial SequenceOligonucleotide primer
71gttaagcgcc tgataggctc 207226DNAArtificial SequenceOligonucleotide
primer 72agccaccccc aggaactgga gataga 267328PRTHomo sapiens 73Ser
Asp Ala Ala Val Asp Thr Ser Ser Glu Ile Thr Thr Lys Asp Leu 1 5 10
15 Lys Glu Lys Lys Glu Val Val Glu Glu Ala Glu Asn 20 25
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