U.S. patent application number 10/424638 was filed with the patent office on 2003-09-18 for replication deficient adenoviral tnf vector.
This patent application is currently assigned to GenVec, Inc.. Invention is credited to Brough, Douglas E., King, C. Richter, Kovesdi, Imre, Schaible, Jasper J..
Application Number | 20030175245 10/424638 |
Document ID | / |
Family ID | 24420637 |
Filed Date | 2003-09-18 |
United States Patent
Application |
20030175245 |
Kind Code |
A1 |
Brough, Douglas E. ; et
al. |
September 18, 2003 |
Replication deficient adenoviral TNF vector
Abstract
An adenoviral vector comprising (a) an adenoviral genome
deficient in the E4 region of the adenoviral genome, (b) a nucleic
acid sequence coding for TNF, and (c) a radiation inducible
promoter operably linked to the nucleic acid sequence coding for
TNF. This invention also provides an adenoviral vector comprising
(a) an adenoviral genome deficient in the E4 region of the
adenoviral genome, (b) a nucleic acid sequence coding for TNF, and
(c) a spacer element of at least 15 base pairs in the E4 region of
the adenoviral genome. A method of producing an adenoviral vector
and a method of treating a tumor or cancer in a host comprising
administering an anti-cancer or anti-tumor effective amount of the
adenoviral vector of the present invention also is provided.
Inventors: |
Brough, Douglas E.;
(Gaithersburg, MD) ; King, C. Richter;
(Washington, DC) ; Kovesdi, Imre; (Rockville,
MD) ; Schaible, Jasper J.; (San Diego, CA) |
Correspondence
Address: |
LEYDIG VOIT & MAYER, LTD
TWO PRUDENTIAL PLAZA, SUITE 4900
180 NORTH STETSON AVENUE
CHICAGO
IL
60601-6780
US
|
Assignee: |
GenVec, Inc.
Gaithersburg
MD
|
Family ID: |
24420637 |
Appl. No.: |
10/424638 |
Filed: |
April 25, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10424638 |
Apr 25, 2003 |
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09604694 |
Jun 27, 2000 |
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6579522 |
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Current U.S.
Class: |
424/93.2 ;
435/235.1; 435/456 |
Current CPC
Class: |
A61K 38/191 20130101;
C12N 2810/40 20130101; A61P 9/10 20180101; A61P 35/00 20180101;
A61K 48/00 20130101; A61P 43/00 20180101; C12N 15/86 20130101; C12N
2710/10343 20130101; A61P 11/00 20180101; A61P 11/06 20180101 |
Class at
Publication: |
424/93.2 ;
435/235.1; 435/456 |
International
Class: |
A61K 048/00; C12N
007/00; C12N 015/861 |
Claims
What is claimed is:
1. An adenoviral vector comprising (a) an adenoviral genome
deficient in the E4 region of the adenoviral genome, and optionally
deficient in the E1 region, the E2A region, and/or the E3 region of
the adenoviral genome, (b) a nucleic acid sequence coding for TNF,
and (c) a radiation inducible promoter operably linked to the
nucleic acid sequence coding for TNF.
2. The adenoviral vector of claim 1, wherein the adenoviral genome
is deficient in the E1 region of the adenoviral genome.
3. The adenoviral vector of claim 2, wherein the nucleic acid
sequence coding for TNF is located in the E1 region of the
adenoviral genome.
4. The adenoviral vector of claim 3, wherein the TNF is
TNF-.alpha..
5. The adenoviral vector of claim 3, wherein the nucleic acid
sequence coding for TNF further comprises a
transcription-terminating region.
6. The adenoviral vector of claim 1, wherein the radiation
inducible promoter is Egr-1.
7. The adenoviral vector of claim 2, wherein the adenoviral vector
is deficient in the E3 region of the adenoviral genome.
8. The adenoviral vector of claim 7, wherein the adenoviral genome
is deficient in the entire E3 region.
9. The adenoviral vector of claim 1, wherein the adenoviral genome
is deficient in the entire coding region of the E4 region.
10. The adenoviral vector of claim 9, wherein the coding region of
the E4 region of the adenoviral genome has been replaced with a
spacer element having at least 15 base pairs.
11. The adenoviral vector of claim 10, wherein the spacer element
is transcriptionally inert.
12. The adenoviral vector of claim 11, wherein the spacer element
comprises a polyadenylation sequence that is non-native to the E4
region of the adenoviral genome.
13. The adenoviral vector of claim 12, wherein the spacer element
comprises an SV40 polyadenylation sequence.
14. The adenoviral vector of claim 10, wherein the viral production
level of the adenoviral vector is greater than the viral production
level of the adenoviral vector without the spacer element.
15. The adenoviral vector of claim 1, wherein the adenoviral genome
is a serotype-5 adenoviral genome.
16. A replication competent adenovirus-free stock of the adenoviral
vector of claim 1.
17. A host cell comprising the adenoviral vector of claim 1.
18. An adenoviral vector comprising (a) an adenoviral genome
deficient in the E4 region of the adenoviral genome, and optionally
deficient in the E1 region, the E2A region, and/or the E3 region of
the adenoviral genome, (b) a nucleic acid sequence coding for a TNF
which is secreted from a cell infected with the adenoviral vector,
and (c) a spacer element of at least 15 base pairs in the E4 region
of the adenoviral genome.
19. The adenoviral vector of claim 18, wherein the adenoviral
genome is deficient in the E1 region of the adenoviral genome.
20. The adenoviral vector of claim 18, wherein the spacer element
is transcriptionally inert.
21. The adenoviral vector of claim 20, wherein the spacer element
comprises a polyadenylation sequence that is non-native to the E4
region of the adenoviral genome.
22. The adenoviral vector of claim 21, wherein the spacer element
comprises an SV40 polyadenylation sequence.
23. The adenoviral vector of claim 19, wherein the nucleic acid
sequence coding for TNF is located in the E1 region of the
adenoviral genome.
24. The adenoviral vector of claim 23, wherein the TNF is
TNF-.alpha..
25. The adenoviral vector of claim 23, wherein the nucleic acid
sequence coding for TNF further comprises a
transcription-terminating region.
26. The adenoviral vector of claim 18, wherein the adenoviral
vector is deficient in the E3 region of the adenoviral genome.
27. The adenoviral vector of claim 26, wherein the adenoviral
genome is deficient in the entire E3 region.
28. The adenoviral vector of claim 18, wherein the adenoviral
genome is deficient in the entire coding region of the E4
region.
29. The adenoviral vector of claim 18, wherein the viral production
level of the adenoviral vector is greater than the viral production
level of the adenoviral vector without the spacer element.
30. The adenoviral vector of claim 18, wherein the adenoviral
genome is a serotype-5 adenoviral genome.
31. A replication competent adenoviral-free stock of the adenoviral
vector of claim 18.
32. A host cell comprising the adenoviral vector of claim 18.
33. A method of treating a tumor or cancer in a mammal comprising
administering an anti-tumor or anti-cancer effective amount of the
adenoviral vector of claim 1 directly to the tumor or cancer of the
mammal.
34. The method of claim 33, further comprising the administration
of radiation to the host.
35. The method of claim 34, wherein the radiation induces
expression of the nucleic acid sequence coding for TNF to produce a
therapeutic level of TNF in the host.
36. The method of claim 35, wherein the administration of radiation
comprises the use of an internal source of radiation.
37. The method of claim 33, further comprising the administration
of a TNF antagonist to the host.
38. The method of claim 37, wherein the TNF antagonist is at least
one selected from the group comprising soluble TNF receptors and
anti-TNF anti-bodies.
39. A method of treating a tumor or cancer in a mammal comprising
administering an anti-tumor or anti-cancer effective amount of the
adenoviral vector of claim 18 directly to the tumor or cancer of
the mammal.
40. A method of producing an adenoviral vector comprising (a)
providing an adenoviral genome that is deficient in the E4 region
of the adenoviral genome, (b) inserting a nucleic acid sequence
coding for TNF into the adenoviral genome, and (c) inserting a
radiation-inducible promoter into the adenoviral genome such that
it is operably linked to the nucleic acid sequence coding for
TNF.
41. The method of claim 40, wherein the adenoviral genome comprises
a spacer element having at least 15 base pairs in the E4 region of
the adenoviral genome.
42. The method of claim 40, wherein the radiation inducible
promoter is Egr-1.
43. The adenoviral vector of claim 1, wherein the nucleic acid
sequence encodes a TNF which is secreted from a cell infected with
the adenoviral vector.
44. The adenoviral vector of claim 1, wherein the nucleic acid
sequence coding for TNF comprises SEQ ID NO: 2.
45. The adenoviral vector of claim 18, wherein the nucleic acid
sequence coding for a TNF which is secreted from a cell infected
with the adenoviral vector comprises SEQ ID NO: 2.
46. The method of claim 33, wherein the adenoviral vector is
administered to a tumor in a mammal.
47. The method of claim 46, wherein the mammal is a human.
48. The method of claim 39, wherein the adenoviral vector is
administered to a tumor in a mammal.
49. The method of claim 48, wherein the mammal is a human.
50. A pharmaceutical composition comprising the adenoviral vector
of claim 1 and a pharmaceutically acceptable carrier, wherein the
pharmaceutical composition does not contain replication-competent
adenoviruses.
51. A pharmaceutical composition comprising the adenoviral vector
of claim 18 and a pharmaceutically acceptable carrier, wherein the
pharmaceutical composition does not contain replication-competent
adenoviruses.
Description
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
[0001] This patent application is a divisional of U.S. patent
application Ser. No. 09/604,694, filed Jun. 27, 2000.
TECHNICAL FIELD OF THE INVENTION
[0002] This invention pertains to a replication deficient
adenoviral vector comprising a nucleic acid sequence coding for
tumor necrosis factor (TNF), as well as a method of constructing
and using such vector.
BACKGROUND OF THE INVENTION
[0003] Tumor necrosis factor (TNF), especially TNF-.alpha., is
well-known for its anti-tumor effects and ability to act
synergistically with radiation therapy. For example, certain
replication deficient adenoviral vectors comprising the TNF-.alpha.
gene have been used in conjunction with radiation therapy to treat
tumors in animals with some success (e.g., Hallahan et al., Nat.
Med., 1, 786-91 (1995)). The use of TNF as an
anti-cancer/anti-tumor agent, however, has been limited by its
severe systemic effects.
[0004] There remains a need for replication deficient TNF
adenoviral vectors that have greater flexibility in their
construction and use, and can provide greater success in the
treatment of a tumor or cancer. The present invention provides such
a vector, as well as a method of constructing such vector, and a
therapeutic method involving the use of such vector.
BRIEF SUMMARY OF THE INVENTION
[0005] The present invention provides an adenoviral vector
comprising (a) an adenoviral genome deficient in the early growth-4
(E4) region of the adenoviral genome, (b) a nucleic acid sequence
coding for TNF, and (c) a radiation inducible promoter operably
linked to the nucleic acid sequence coding for TNF. This invention
also provides an adenoviral vector comprising (a) an adenoviral
genome deficient in the E4 region of the adenoviral genome, (b) a
nucleic acid sequence coding for TNF, and (c) a spacer element of
at least 15 base pairs in the E4 region of the adenoviral
genome.
[0006] A method of producing an adenoviral vector also is provided
by the present invention comprising (a) providing an adenoviral
genome that is deficient in the E4 region of the adenoviral genome,
(b) inserting a nucleic acid sequence coding for TNF into the
adenoviral genome, and (c) inserting a radiation-inducible promoter
into the adenoviral genome such that it is operably linked to the
nucleic acid sequence coding for TNF. This invention further
provides a method of treating a tumor or cancer in a host
comprising administering an anti-cancer or anti-tumor effective
amount of the adenoviral vector of the present invention to a host
in need thereof.
[0007] The present invention may best be understood with reference
to the accompanying drawings and in the following detailed
description of the preferred embodiments.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1A is a schematic representation of a vector containing
an unmodified adenoviral genome.
[0009] FIG. 1B is a schematic representation of a vector containing
a modified adenoviral genome and the genetic elements in accordance
with the present invention.
[0010] FIG. 2 is a plot of tumor volume against time for a tumor
treated in a variety of manners, including with the adenoviral
vector of the present invention.
[0011] FIG. 3 is another plot of tumor volume against time for a
tumor treated in a variety of manners, including with the
adenoviral vector of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0012] The present invention provides an adenoviral vector
comprising (a) an adenoviral genome deficient in the E4 region of
the adenoviral genome, (b) a nucleic acid sequence coding for TNF,
and (c) a radiation inducible promoter operably linked to the
nucleic acid sequence coding for TNF. The present invention also
provides an adenoviral vector comprising (a) an adenoviral genome
deficient in the E4 region of the adenoviral genome, (b) a nucleic
acid sequence coding for TNF, and (c) a spacer element of at least
15 base pairs in the E4 region of the adenoviral genome.
Adenoviral Genome
[0013] Any subtype, mixture of subtypes, or chimeric adenovirus can
be used as the source of the viral genome for generation of an
adenoviral vector in conjunction with the present invention.
Preferably the genome of a human serotype adenovirus is used, such
as a type 2 (Ad2) or type 5 (Ad5) adenoviral genome. Although any
suitable adenoviral genome can be used in conjunction with the
present invention, the Ad5 adenoviral genome is most preferred, and
the present invention is described further herein with respect to
the Ad5serotype.
[0014] The adenoviral genome used in conjunction with the present
invention is desirably replication deficient. A deficiency in a
gene, gene function, or gene or genomic region, as used herein, is
defined as a deletion of sufficient genetic material of the viral
genome to impiar or obliterate the function of the gene whose
nucleic acid sequence was deleted in whole or in part and to
provide room in, or capacity of, the viral genome for the insertion
of a nucleic acid sequence that is foreign to the viral genome.
Such a deficiency can be in a gene or genome region essential or
unessential for propagation of the adenoviral vector in a
non-complementing cellular host. A deficiency in an adenoviral
genome region essential for such propagation (e.g., early region 1
(E1), early region 2A (E2A), early region 2B (E2B), early region 4
(E4), late region 1 (L1), late region 2 (L2), late region 3 (L3),
late region 4 (L4), and late region 5 (L5)) renders an adenoviral
vector based on that adenoviral genome replication deficient.
[0015] The adenoviral vector of the present invention desirably is
multiply replication deficient, i.e., it is deficient in at least
two genome regions required for viral propagation in a
non-complementing cellular host (i.e., viral replication in vitro).
Such regions include the E1, E2, E4, or L1-L5 regions. Even though
the E1 region can be considered as consisting of early region 1A
(E1A) and early region 1B (E1B), a deficiency in either or both of
the E1A and/or E1 B regions is considered as a single deficiency in
the context of the present invention. In addition, such a vector
can be deficient in one or more regions that are not required for
viral propagation, e.g., the vectors can be additionally deficient
in early region 3 (E3).
[0016] The present inventive adenoviral vector desirably is
deficient in the E1 and E4 regions, preferably with the entire
coding region of the E4 region having been deleted from the
adenoviral vector. In the context of the present invention, the
coding region refers to the portion or portions of a genomic region
that encode a protein product (e.g., the open reading frames (ORFs)
of the E4 region). Thus, a vector in which the entire coding region
of the E4 region of the adenoviral genome has been deleted is
lacking all of the ORFs of that region. The E4 region of the
present inventive adenoviral vector preferably retains the native
E4 promoter, polyadenylation sequence, and/or the right-side
inverted terminal repeat (ITR).
[0017] The adenoviral vector of the present invention also can be
deficient in one or more additional regions required for viral
propagation (especially other early regions required for viral
propagation, such as the E2A and/or E2B regions). More preferably,
the E3 region of the adenoviral genome also is removed. Thus,
preferred configurations of the present inventive adenoviral vector
include (a) E1.sup.- E4.sup.-, (b) E1.sup.- E2A.sup.- E4.sup.-, and
(c) E2A.sup.- E4.sup.- adenoviral vectors, any of which also can be
E3.sup.-.
[0018] FIG. 1A provides a schematic representation of the Ad5
genome. FIG. 1B is a schematic representation of an exemplary
adenoviral vector, which figure shows, by comparison to the
unaltered Ad5 adenoviral genome represented by FIG. 1A, that (i)
the E1region (10) has been replaced by a radiation inducible
promoter (14), a nucleic acid sequence encoding TNF (15), and a
polyadenylation sequence (16), (ii) the E3 region (11) has been
deleted (17), and (iii) the coding region of the E4 region (12) has
been replaced by a spacer element (18), thereby creating a vector
deficient in the E1, E3, and E4 regions.
[0019] If the adenoviral vector of the present invention is
deficient in the E2A region, the vector preferably further
comprises a portion of the E2A region of the adenoviral genome in
the E2A deficient region, which is less than about 230 base pairs
in length. Generally, the E2A region of the adenovirus codes for
DBP (DNA binding protein), a polypeptide required for DNA
replication. DBP is composed of 473 to 529 amino acids depending on
the viral serotype. It is believed that DBP is an asymmetric
protein that exists as a prolate ellipsoid consisting of a globular
Ct with an extended Nt domain. Studies indicate that the Ct domain
is responsible for DBP's ability to bind to nucleic acids, bind to
zinc, and function in DNA synthesis at the level of DNA chain
elongation. However, the Nt domain is believed to function in late
gene expression at both transcriptional and post-transcriptional
levels, is responsible for efficient nuclear localization of the
protein, and also may be involved in enhancement of its own
expression. Deletions in the Nt domain between amino acids 2 to 38
have indicated that this region is important for DBP function
(Brough et al., Virology, 196, 269-281 (1993)). While deletions in
the E2A region coding for the Ct region of the DBP have no effect
on viral propagation, deletions in the E2A region which code for
amino acids 2 to 38 of the Nt domain of the DBP impair viral
propagation. Therefore, it is preferable that any multiply
replication deficient adenoviral vector contain this portion of the
E2A region of the adenoviral genome.
[0020] In particular, for example, the desired portion of the E2A
region to be retained is that portion of the E2A region of the
adenoviral genome which is defined by the 5' end of the E2A region,
specifically, positions Ad5(23816) to Ad5(24032) of the E2A region
of the adenoviral genome of serotype Ad5. This portion of the
adenoviral genome desirably is included in the adenoviral vector
because it is not complemented in current E2A cell lines, and in
its absence the requisite levels of viral propagation and fiber
expression cannot be obtained in complementing cell lines.
[0021] The present invention is not limited to adenoviral vectors
that are deficient in gene functions only in the early region of
the genome. Also included are adenoviral vectors that are deficient
in the early and late regions of the genome, as well as vectors in
which essentially the entire genome has been removed, in which case
it is preferred that at least either the viral ITRs and some of the
promoters or the viral ITRs and a packaging signal are left intact.
One of ordinary skill in the art will appreciate that the larger
the region of the adenoviral genome that is removed, the larger the
piece of exogenous nucleic acid sequence that can be inserted into
the genome. For example, given that the adenoviral genome is 36 kb,
by leaving the viral ITRs and some of the promoters intact, the
capacity of the adenovirus is approximately 35 kb. Alternatively,
one could generate a multiply deficient adenoviral vector that
contains only the ITR and a packaging signal. This could then
effectively allow for expression of 37-38 kb of foreign nucleic
acid sequence from this vector. Of course, the inclusion of a
spacer element in any or all of the deficient adenoviral regions
will decrease the capacity of the adenoviral vector in size
corresponding with the size of the spacer element sequence.
Nucleic Acid Sequence Encoding TNF
[0022] Preferably, the vector comprises at least one expression
cassette which includes (i.e., comprises) a nucleic acid sequence
coding for TNF. Nucleic acid sequences encoding TNF include nucleic
acid sequences encoding any member of the TNF family of proteins.
The adenoviral vector of the present invention preferably comprises
a nucleic acid sequence coding for TNF-.alpha.. A nucleic acid
sequence coding for TNF is described in detail in U.S. Pat. No.
4,879,226, which discloses a nucleic acid sequence encoding a
"human TNF, in mature form, secreted from host cells" (see column
7, lines 26-28), as set forth in SEQ ID NO: 2.
[0023] The nucleic acid sequence coding for TNF is preferably
located in the E1 region (e.g., replaces the E1 region in whole or
in part, preferably in whole) of the adenoviral genome. For
example, the E1 region can be replaced by a promoter-variable
expression cassette comprising a nucleic acid seqence encoding TNF.
The term "expression cassette," as used herein, refers to any
nucleic acid sequence that can be inserted into the adenoviral
genome to produce a foreign gene product. For example, as shown in
FIGS. 1A and 1B, the E1region (10) can be replaced by an expression
cassette (13) comprising a radiation inducible promoter (14), a
nucleic acid sequence encoding TNF (15), and a polyadenylation
sequence (16). In addition to the expression cassette comprising
the nucleic acid sequence encoding TNF, the vector can comprise
other expression cassettes containing nucleic acid sequences
encoding other products, which cassettes can replace any of the
deleted regions of the adenoviral genome. The insertion of an
expression cassette into the adenoviral genome (e.g., the E1 region
of the genome) can be facilitated by known methods, for example, by
the introduction of a unique restriction site at a given position
of the adenoviral genome.
[0024] Preferably, the nucleic acid sequence encoding TNF further
comprises a transcription-terminating region such as a
polyadenylation sequence located 3' of the region encoding TNF. Any
suitable polyadenylation sequence can be used, including a
synthetic optimized sequence, as well as the polyadenylation
sequence of BGH (Bovine Growth Hormone), polyoma virus, TK
(Thymidine Kinase), EBV (Epstein Barr Virus), and the
papillomaviruses, including human papillomaviruses and BPV (Bovine
Papilloma Virus). A preferred polyadenylation sequence is the SV40
(Human Sarcoma Virus-40) polyadenylation sequence.
[0025] Preferably, the nucleic acid sequence encoding TNF is
operably linked to (i.e., under the transcriptional control of) one
or more promoter and/or enhancer elements, for example, as part of
a promoter variable expression cassette. Techniques for operably
linking sequences together are well known in the art. Any suitable
promoter or enhancer sequence can be used in conjunction with the
present invention. Suitable promoters and enhancer sequences are
generally known in the art. Preferred vectors according to the
present invention comprise a radiation-inducible promoter operably
linked to a nucleic acid sequence encoding TNF. The use of a
radiation inducible promoter provides control over transcription of
the foreign gene product, for example, by the administration of
radiation to a cell or host comprising the adenoviral vector. Any
suitable radiation inducible promoter can be used in conjunction
with the present invention. Suitable radiation inducible promoters
are generally known in the art. A preferred radiation inducible
promoter for use in conjunction with the present invention is the
early growth region-1(Egr-1) promoter, specifically the CArG domain
of the Egr-1promoter. The Egr-1 promoter has been described in
detail in U.S. Pat. No. 5,206,152 and International Patent
Application WO 94/06916. The promoter can be introduced into the
foreign genome by methods known in the art, for example, by the
introduction of a unique restriction site at a give region of the
genome. Alternatively, the promoter can be inserted as part of the
expression cassette comprising the nucleic acid sequence coding for
TNF.
Spacer Element
[0026] The present inventive adenoviral vector, when multiply
replication deficient, preferably includes a spacer element to
provide viral growth in a complementing cell line similar to that
achieved by singly replication deficient adenoviral vectors,
particularly a singly replication deficient E1.sup.- adenoviral
vector. In the absence of a spacer, production of fiber protein
and/or viral growth of the multiply replication deficient
adenoviral vector is reduced by comparison to that of a singly
replication deficient adenoviral vector. However, inclusion of the
spacer in at least one of the deficient adenoviral regions,
preferably the E4 region as shown in FIG. 1B (18), counteracts this
defect in growth and fiber expression.
[0027] In the preferred E4.sup.- adenoviral vector of the present
invention wherein the L5 fiber region is retained, the spacer is
desirably located between the L5 fiber region and the right-side
ITR. More preferably, in such an adenoviral vector, the E4
polyadenylation sequence alone or, most preferably, in combination
with another sequence, exists between the L5 fiber region and the
right-side ITR, so as to sufficiently separate the retained L5
fiber region from the right-side ITR, such that viral production of
such a vector approaches that of a singly replication deficient
adenoviral vector, particularly a singly replication deficient
E1.sup.- adenoviral vector. The use of a spacer in an adenoviral
vector is described in U.S. Pat. No. 5,851,806.
[0028] As the function of the replication deficient region of the
genome can be provided by a complementing cell line, the spacer
element does not need to provide the deficient function and can be
any sequence. Thus, the spacer element is limited only by the size
of the insert that the vector will accommodate. The spacer element
can be of any suitable size, desirably at least about 15 base pairs
(e.g., between about 15 base pairs and about 12,000 base pairs),
preferably about 100 base pairs to about 10,000 base pairs, more
preferably about 500 base pairs to about 8,000 base pairs, even
more preferably about 1,500 base pairs to about 6,000 base pairs,
and most preferably about 2,000 to about 3,000 base pairs.
[0029] The spacer element can contain any sequence or sequences
which are of the desired length. The spacer element sequence can be
coding or non-coding and native or non-native with respect to the
adenoviral genome, but does not restore the replication function to
the deficient region. The spacer element can also contain a
promoter-variable expression cassette. More preferably, the spacer
element comprises an additional polyadenylation sequence and/or a
foreign gene. Preferably, in the case of a spacer element inserted
into a region deficient for E4, both the E4 polyadenylation
sequence and the E4 promoter of the adenoviral genome or any other
(cellular or viral) promoter remain in the vector. In such an
embodiment, the spacer element is located between the E4
polyadenylation site and the E4 promoter, or, if the E4 promotor is
not present in the vector, the spacer element is proximal to the
right-side ITR.
[0030] The spacer element can comprise any suitable polyadenylation
sequence. Examples of suitable polyadenylation sequences include
synthetic optimized sequences, as well as the polyadenylation
sequences of BGH (Bovine Growth Hormone), polyoma virus, TK
(Thymidine Kinase), EBV (Epstein Barr Virus), and the
papillomaviruses, including human papillomaviruses and BPV (Bovine
Papilloma Virus). Preferably, particularly in the E4 deficient
region, the spacer element includes an SV40 (Human Sarcoma
Virus-40) polyadenylation sequence. The SV40 polyadenylation
sequence allows for higher virus production levels of multiply
replication deficient adenoviral vectors.
[0031] A foreign gene also can function as the spacer element in
the E4 deficient region of the adenoviral genome. The foreign gene
is limited only by the size of the fragment the vector can
accommodate and can be any suitable gene. Examples of suitable
foreign genes include marker gene sequences such as pGUS, secretory
alkaline phosphatase, luciferase, B-galactosidase, and human
anti-trypsin; therapeutic genes; potential immune modifiers such as
B3-19K, E3-14.7, ICP47, fas ligand gene, and CTLA4 genes;
biologically inactive sequences (e.g. sequences that will not be
transcribed to produce a product or which encode a defective or
biologically inactive product), and other innocuous sequences such
as the glucuronidase gene.
Vector Construction
[0032] The present invention provides a method of producing an
adenoviral vector comprising (a) providing an adenoviral genome
that is deficient in the E4 region of the adenoviral genome, (b)
inserting a nucleic acid sequence coding for TNF into the
adenoviral genome, and (c) inserting a radiation-inducible promoter
into the adenoviral genome such that it is operably linked to the
nucleic acid sequence coding for TNF. The present invention also
provides a method of producing an adenoviral vector comprising (a)
providing an adenoviral genome that is deficient in the E4 region
of the adenoviral genome, (b) inserting a nucleic acid sequence
coding for TNF into the adenoviral genome, and (c) inserting a
spacer element into the E4 region of the adenoviral genome. As
those of ordinary skill in the art will appreciate, the method
provided by the present invention can include other steps or
elements, such as the insertion of other nucleic acid sequences
into, or deletion of such sequences from, the adenoviral genome
used to provide the adenoviral vector. Furthermore, the various
aspects of the present inventive method (e.g., the adenoviral
genome, nucleic acid sequences coding for TNF, radiation inducible
promoter, spacer element, etc.) are as previously described herein
with respect to the adenoviral vector of the present invention.
[0033] The present inventive method of producing an adenoviral
vector can be carried out using techniques known to those of
ordinary skill in the art. In general, virus vector construction
relies on the high level of recombination between adenoviral
nucleic acid sequences in a cell. Two or three separate adenoviral
nucleic acid sequences (e.g., DNA fragments), containing regions of
similarity (or overlap) between sequences and constituting the
entire length of the genome, are transfected into a cell. The host
cell's recombination machinery constructs a full-length viral
vector genome by recombining the aforementioned seqences. Other
suitable procedures for constructing viruses containing alterations
in various single regions have been previously described (Berkner
et al., Nucleic Acids Res., 12, 925-941 (1984); Berkner et al.,
Nucleic Acids Res., 11, 6003-6020 (1983); Brough et al., Virol.,
190, 624-634 (1992)) and can be used to construct multiply
deficient viruses; yet other suitable procedures include, for
example, in vitro recombination and ligation.
[0034] A preferred method of constructing the present inventive
adenoviral vector first involves constructing the necessary
deletions or modifications (such as adding a spacer element to a
deleted region) of a particular region of the adenoviral genome.
Such modifications can be performed, for example, in a plasmid
cassette using standard molecular biological techniques. The
altered nucleic acid sequence (containing the deletion or
modification) then is moved into a much larger plasmid that
contains up to one-half of the adenovirus genome to provide a base
plasmid comprising the modified adenoviral genome. The next step is
to insert an expression cassette into a desired region of the
modified adenoviral genome. The expression cassette can be provided
by standard methods known in the art, for example, by isolating the
cassette from a plasmid. The isolated cassette then can be
transfected with the plasmid DNA (containing the modified
adenoviral genome) into a recipient cell. The plasmid is,
optionally, linearized prior to transfection by digestion with a
suitable restriction enzyme to facilitate the insertion of the
expression cassette at a desired position in the adenoviral genome.
Selection of a suitable restriction enzyme is well within the skill
of the ordinary artisan. The two pieces of DNA recombine to form a
plasmid comprising the modified adenoviral genome and the
expression cassette. The plasmid is isolated from the host cell and
introduced into recipient cell that complements for the missing
viral functions of the recombined viral genome to produce the
adenoviral vector comprising the modified viral genome and the
expression cassette. The vector can be further modified by
alteration of the ITR and/or packaging signal.
Complementing Cell Lines
[0035] Complementing cell lines for propagation or growth of the
present inventive replication deficient adenoviral vectors are
known and described in detail in U.S. Pat. No. 5,851,806 and Brough
et al., Virol., 70, 6497-6501 (1996). The preferred cell lines are
characterized in complementing for at least one gene function of
the gene functions comprising the E1, E2, and E4 regions of the
adenoviral genome. Other cell lines include those that complement
adenoviral vectors that are deficient in at least one gene function
from the gene functions comprising the late regions, those that
complement for a combination of early and late gene functions, and
those that complement for all adenoviral functions. One of ordinary
skill in the art will appreciate that the cell line of choice is
one that specifically complements for those functions that are
missing from the recombinant replication deficient adenoviral
vector of interest and that are generated using standard molecular
biological techniques. The cell lines are further characterized in
that they contain the complementing genes in a non-overlapping
fashion, which minimizes, and practically eliminates, the
possibility of the vector genome recombining with the cellular DNA.
Accordingly, replication competent adenoviruses are not present in
vector stocks, which are, therefore, suitable for certain
therapeutic purposes, especially gene therapy purposes. This also
avoids the replication of the adenoviruses in non-complementing
cells.
[0036] The complementing cell line must be one that is capable of
expressing the products of the deficient adenoviral gene functions
at the appropriate level for those products in order to generate a
high titer stock of recombinant adenoviral vector. For example, it
is necessary to express the E2A product, DBP, at stoichiometric
levels, i.e., relatively high levels, for adenoviral DNA
replication, but the E2B product, Ad pol, is necessary at only
catalytic levels, i.e., relatively low levels, for adenoviral DNA
replication. Not only must the level of the product be appropriate,
the temporal expression of the product must be consistent with that
seen in normal viral infection of a cell to assure a high titer
stock of recombinant adenoviral vector. For example, the components
necessary for viral DNA replication must be expressed before those
necessary for virion assembly. In order to avoid cellular toxicity,
which often accompanies high levels of expression of the viral
products, and to regulate the temporal expression of the products,
inducible promoter systems are used. For example, the sheep
metallothionine inducible promoter system can be used to express
the complete E4 region, the open reading frame 6 of the E4 region,
and the E2A region. Other examples of suitable inducible promoter
systems include, but are not limited to, the bacterial lac operon,
the tetracycline operon, the T7 polymerase system, and combinations
and chimeric constructs of eukaryotic and prokaryotic transcription
factors, repressors, and other components. Where the viral product
to be expressed is highly toxic, it is desirable to use a bipartite
inducible system, wherein the inducer is carried in a viral vector
and the inducible product is carried within the chromatin of the
complementing cell line. Repressible/inducible expression systems,
such as the tetracycline expression system and lac expression
system also can be used.
Methods of Use
[0037] The present invention provides a method of treating a tumor
or cancer in a host comprising administering an anti-cancer or
anti-tumor effective amount of the adenoviral vector of the present
invention to a host in need thereof.
[0038] One skilled in the art will appreciate that suitable methods
of administering a replication deficient adenoviral vector of the
present invention to an animal for therapeutic or prophylactic
purposes, e.g., gene therapy, vaccination, and the like (see, for
example, Rosenfeld et al., Science, 252, 431-434 (1991), Jaffe et
al., Clin. Res., 39(2), 302A (1991), Rosenfeld et al., Clin. Res.,
39(2), 311A (1991), Berkner, BioTechniques, 6, 616-629 (1988)), are
available, and, although more than one route can be used to
administer the vector, a particular route can provide a more
immediate and more effective reaction than another route.
[0039] The present invention provides a pharmaceutical composition
comprising the adenoviral vector of the present invention and a
carrier, especially a pharmaceutically acceptable (e.g., a
physiologically or pharmacologically acceptable) carrier (e.g.,
excipient or diluent). Pharmaceutically acceptable carriers are
well-known to those who are skilled in the art and are readily
available. The choice of carrier will be determined in part by the
particular method used to administer the pharmaceutical
composition. Accordingly, there is a wide variety of suitable
formulations of the pharmaceutical composition of the present
invention. The following formulations and methods are merely
exemplary and are in no way limiting. However, oral, injectable and
aerosol formulations are preferred.
[0040] Formulations suitable for oral administration can consist of
(a) liquid solutions, such as an effective amount of the compound
dissolved in diluents, such as water, saline, or orange juice; (b)
capsules, sachets or tablets, each containing a predetermined
amount of the active ingredient, as solids or granules; (c)
suspensions in an appropriate liquid; and (d) suitable emulsions.
Tablet forms can include one or more of lactose, mannitol, corn
starch, potato starch, microcrystalline cellulose, acacia, gelatin,
colloidal silicon dioxide, croscarmellose sodium, talc, magnesium
stearate, stearic acid, and other excipients, colorants, diluents,
buffering agents, moistening agents, preservatives, flavoring
agents, and pharmacologically compatible excipients. Lozenge forms
can comprise the active ingredient in a flavor, usually sucrose and
acacia or tragacanth, as well as pastilles comprising the active
ingredient in an inert base, such as gelatin and glycerin, or
sucrose and acacia, emulsions, gels, and the like containing, in
addition to the active ingredient, such excipients as are known in
the art.
[0041] The vectors of the present invention, alone or in
combination with other suitable components, can be made into
aerosol formulations to be administered via inhalation. These
aerosol formulations can be placed into pressurized acceptable
propellants, such as dichlorodifluoromethane, propane, nitrogen,
and the like. They also can be formulated as pharmaceuticals for
non-pressured preparations, such as in a nebulizer or an
atomizer.
[0042] Formulations suitable for parenteral administration include
aqueous and non-aqueous, isotonic sterile injection solutions,
which can contain anti-oxidants, buffers, bacteriostats, and
solutes that render the formulation isotonic with the blood of the
intended recipient, and aqueous and non-aqueous sterile suspensions
that can include suspending agents, solubilizers, thickening
agents, stabilizers, and preservatives. The formulations can be
presented in unit-dose or multi-dose sealed containers, such as
ampules and vials, and can be stored in a freeze-dried
(lyophilized) condition requiring only the addition of the sterile
liquid excipient, for example, water, for injections, immediately
prior to use. Extemporaneous injection solutions and suspensions
can be prepared from sterile powders, granules, and tablets of the
kind previously described.
[0043] Additionally, the vectors employed in the present invention
can be made into suppositories by mixing with a variety of bases
such as emulsifying bases or watersoluble bases. Formulations
suitable for vaginal administration can be presented as pessaries,
tampons, creams, gels, pastes, foams, or spray formulas containing,
in addition to the active ingredient, such carriers as are known in
the art to be appropriate.
[0044] The dose administered to an animal, particularly a human, in
the context of the present invention will vary with the particular
adenoviral vector, the composition containing the adenoviral
vector, the method of administration, and the particular site and
organism being treated. The dose should be sufficient to effect a
desirable response, e.g., therapeutic or prophylactic response,
within a desirable time frame.
[0045] The present method of treating a tumor or cancer in a host
further can comprise the administration (i.e., pre-administration,
co-administration, and/or post-administration) of other treatments
and/or agents to modify (e.g., enhance) the effectiveness thereof.
For example, an adenoviral vector of the present invention,
particularly a vector comprising a nucleic acid sequence coding for
TNF that is operably linked to a radiation inducible promoter, can
be administered in conjunction with the administration of
radiation. The radiation can be administered in any suitable
manner, for example, by exposure to an external source of radiation
(e.g., infrared radiation), or through the use of an internal
source of radiation (e.g., through the chemical or surgical
administration of a source of radiation). For instance, the
adenoviral vector of the present invention can be used in
conjunction with brachytherapy, wherein a radioactive source is
placed (i.e., implanted) in or near a tumor to deliver a high,
localized dose of radiation. Radiation is desirably administered in
a dose sufficient to induce the production of a therapeutic level
of TNF in the host.
[0046] The method of the present invention, additionally or
alternatively to the administration of radiation, further can
comprise the administration of other substances which locally or
systemically alter (i.e., diminish or enhance) the effect of TNF in
vivo. For example, substances that diminish the systemic effect of
TNF can be used to control the level of systemic toxicity expressed
in a host. Likewise, substances that enhance the local effect of
TNF can be used to reduce the level of TNF required to produce a
prophylactic or therapeutic effect in a host. Such substances
include TNF antagonists, for example, soluble TNF receptors or
anti-TNF antibodies, and TNF agonists. Other suitable antagonists,
agonists, and other substances that alter the effect of TNF are
available and generally known in the art.
[0047] The replication deficient adenoviral vectors of the present
invention also have utility in vitro. For example, they can be used
to study adenoviral gene function and assembly, the production of
TNF, or the expression of other foreign nucleic acid sequences in a
suitable target cell. One of ordinary skill can identify a suitable
target cell by selecting one that can be transfected by the
adenoviral vector, resulting in expression of the thereby inserted
adenoviral nucleic acid sequence complement. Preferably, a suitable
target cell is selected that has receptors for attachment and
penetration of adenovirus into a cell. Such cells include, but are
not limited to, those originally isolated from any mammal. Once the
suitable target cell has been selected, the target cell is
contacted with an adenoviral vector of the present invention,
thereby effecting transfection or infection, respectively.
Expression, toxicity, and other parameters relating to the
insertion and activity of the nucleic acid sequence encoding TNF,
or other foreign nucleic acid sequences, in the target cell then is
measured using conventional methods well known in the art. For
example, the adenoviral vector of the present invention can be used
to study the interaction and targeting of TNF with respect to other
cellular molecules. In so doing, researchers can learn and
elucidate the phenomenology concerning adenoviral infection as well
as the efficacy and effect of expression of various foreign nucleic
acid sequences introduced by the adenoviral vector in various cell
types that are explanted from various organisms and studied in
tissue culture.
[0048] Moreover, cells explanted or removed from a patient having a
disease that is suitably treated by gene therapy in the context of
the present invention usefully are manipulated in vitro. For
example, cells cultured in vitro from such an individual are placed
in contact with an adenoviral vector of the present invention under
suitable conditions to effect transfection, which are readily
determined by one of ordinary skill in the art. Such contact
suitably results in transfection of the vector into the cultured
cells, where the transfected cells are selected using a suitable
marker and selective culturing conditions. In so doing, using
standard methods to test for vitality of the cells and thus measure
toxicity and to test for presence of gene products of the foreign
nucleic acid sequences of the vector of the present invention and
thus measure expression, the cells of the individual are tested for
compatibility with, expression in, and toxicity of the vector of
the present invention, thereby providing information as to the
appropriateness and efficacy of treatment of the individual with
the vector system so tested. Such explanted and transfected cells,
in addition to serving to test the potential efficacy/toxicity of a
given gene therapy regime, also can be returned to an in vivo
position within the body of the individual. Such cells so returned
to the individual can be returned unaltered and unadorned except
for the in vitro transfection thereof, or encased by or embedded in
a matrix that keeps them separate from other tissues and cells of
the individual's body. Such a matrix can be any suitable
biocompatible material, including collagen, cellulose, and the
like. Of course, alternatively or in addition, preferably after a
positive response to the in vitro test, the transfection can be
implemented in vivo by administration means as detailed
hereinabove.
Further Aspects of the Present Invention
[0049] As those of ordinary skill in the art will appreciate, the
adenoviral vector, and methods involving the same, provided by the
present invention can comprise any combination or permutation of
the elements described herein. A vector having a preferred
configuration of such elements is schematically represented by FIG.
1B. Furthermore, a vector having such configuration of elements is
provided by a vector that comprises, consists essentially of, or
consists of the nucleic acid sequence of SEQ ID NO: 1. However,
many modifications and variations of the present illustrative
nucleic acid sequence are possible. For example, the degeneracy of
the genetic code allows for the substitution of nucleotides
throughout polypeptide coding regions, as well as in the
translational stop signal, without alteration of the encoded
polypeptide coding sequence. Such substitutable sequences can be
deduced from the known amino acid or nucleic acid sequence of a
given gene and can be constructed by conventional synthetic or
site-specific mutagenesis procedures. Synthetic DNA methods can be
carried out in substantial accordance with the procedures of
Itakura et al., Science, 198, 1056-1063 (1977), and Crea et al.,
Proc. Natl. Acad. Sci. USA, 75, 5765-5769 (1978). Site-specific
mutagenesis procedures are described in Maniatis et al., Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y. (2d ed.
1989). Therefore, the present invention is in no way limited to the
nucleic acid sequence specifically exemplified herein. Exemplified
vectors are for gene therapy of tumors and/or cancer and,
therefore, contain and express the TNF gene. However, the vectors
described also can comprise genes used to treat other similar or
different diseases and/or afflictions including, but not limited
to, other chronic lung diseases, such as emphysema, asthma, adult
respiratory distress syndrome, and chronic bronchitis, as well as
coronary heart disease, and other afflictions suitably treated or
prevented by gene therapy, vaccination, and the like. Accordingly,
any gene or nucleic acid sequence can be inserted into the
adenoviral TNF vector.
[0050] The adenoviral vector can be modified in other ways without
departing from the scope and spirit of the present invention. For
example, the coat protein of the present inventive adenoviral
vector can be manipulated to alter the binding specificity or
recognition of the virus for a viral receptor on a potential host
cell. Such manipulations can include deletion of regions of the
fiber, penton, or hexon, insertions of various native or non-native
ligands into portions of the coat protein, and the like.
Manipulation of the coat protein can broaden the range of cells
infected by a viral vector or enable targeting of a viral vector to
a specific cell type.
[0051] For example, the vector can comprise a chimeric coat protein
(e.g., a fiber, hexon or penton protein), which differs from the
wild-type (i.e., native) coat protein by the introduction of a
nonnative amino acid sequence, preferably at or near the carboxyl
terminus. Preferably, the nonnative amino acid sequence is inserted
into or in place of an internal coat protein sequence. The
resultant chimeric viral coat protein is able to direct entry into
cells of the adenoviral vector comprising the coat protein that is
more efficient than entry into cells of an adenoviral vector that
is identical except for comprising a wild-type viral coat protein
rather than the chimeric viral coat protein.
[0052] The chimeric virus coat protein desirably binds a novel
endogenous binding site present on the cell surface. A result of
this increased efficiency of entry is that the adenoviral virus can
bind to and enter numerous cell types which a virus comprising
wild-type coat protein typically cannot enter or can enter with
only a low efficiency.
[0053] Alternatively, the adenoviral vector of the present
invention can comprise a chimeric virus coat protein that is not
selective for a specific type of eukaryotic cell. Such chimeric
coat protein differs from the wild-type coat protein by an
insertion of a normative amino acid sequence into or in place of an
internal coat protein sequence. In a vector comprising a
non-selective chimeric coat protein, the virus coat efficiently
binds to a broader range of eukaryotic cells than a wild-type virus
coat, such as described in International Patent Application WO
97/20051.
[0054] Specificity of binding of an adenovirus to a given cell also
can be adjusted by use of an adenovirus comprising a short-shafted
adenoviral fiber gene, as discussed in U.S. Pat. No. 5,962,311. Use
of an adenovirus comprising a short-shafted adenoviral fiber gene
reduces the level or efficiency of adenoviral fiber binding to its
cell-surface receptor and increases adenoviral penton base binding
to its cell-surface receptor, thereby increasing the specificity of
binding of the adenovirus to a given cell. Alternatively, use of an
adenovirus comprising a short-shafted fiber enables targeting of
the adenovirus to a desired cell-surface receptor by the
introduction of a normative amino acid sequence either into the
penton base or the fiber knob.
[0055] In addition, the ability of a viral vector to recognize a
potential host cell can be modulated without genetic manipulation
of the coat protein. For instance, complexing an adenovirus with a
bispecific molecule comprising a penton base-binding domain and a
domain that selectively binds a particular cell surface binding
site enables one of ordinary skill in the art to target the vector
to a particular cell type.
[0056] Many modifications to a viral vector, specifically an
adenoviral vector, are known in the art. Suitable modifications for
an adenoviral vector include those modifications described in U.S.
Pat. Nos. 5,559,099; 5,731,190; 5,712,136; 5,770,442; 5,846,782;
5,926,311; and 5,965,541 and International Patent Applications WO
96/07734, WO 96/26281, WO 97/20051, WO 98/07865, WO 98/07877, and
WO 98/54346.
[0057] The following examples further illustrate the present
invention and, of course, should not be construed as in any way
limiting its scope. Enzymes referred to in the examples are
available, unless otherwise indicated, from Bethesda Research
Laboratories (BRL), Gaithersburg, Md., New England Biolabs Inc.
(NEB), Beverly, Mass., or Boehringer Mannheim Biochemicals (BMB),
Indianapolis, Ind., and are used in substantial accordance with the
manufacturer's recommendations. Many of the techniques employed
herein are well known to those in the art. Molecular biology
techniques are described in detail in suitable laboratory manuals,
such as Maniatis et al., Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor, N.Y. (2d ed. 1989), and Current Protocols in
Molecular Biology (Ausubel et al., eds. (1987)).
EXAMPLE 1
[0058] The following example demonstrates the preparation of an
adenoviral vector in accordance with the present invention. A first
plasmid (base plasmid) comprising a type-5 adenoviral genome was
prepared having deficiencies in the E1, E3, and E4 regions, wherein
the E3 region was entirely deleted and the E4 region comprised an
SV40 polyadenylation sequence. A second plasmid (donor plasmid) was
prepared comprising an expression cassette having an Egr-1
radiation-inducible promoter operably linked to a nucleic acid
sequence encoding TNF-.alpha.. The donor plasmid was digested with
Drd-I restriction endonuclease, and a nucleic acid segment of
approximately 6.5 kbp comprising the Egr-I/TNF-.alpha. expression
cassette was isolated by gel electrophoresis. The base plasmid was
digested using Swa-I restriction endonuclease, and the linearized
plasmid was de-phosphorylated. Competent E. coli BJIq cells were
transformed with 50 ng of the 6.5 kbp (approx.) donor plasmid
fragment and 50 ng of the linearized base plasmid. The transformed
cells were plated on luria/SOC broth plates containing 50
.mu.g/.mu.l of kanamycin and 50 .mu.g/.mu.l tetracycline. The
resulting colonies were screened for recombinants, and the positive
recombinants were grown on luria broth/kanamycin plates.
[0059] The recombinant plasmid DNA was isolated from the
transformed E. coli BJIq cells by standard techniques. Using
standard procedures, the isolated plasmid DNA was linearized by
digestion with Pac-I restriction endonuclease, and the linearized
plasmid DNA was transfected in 293-ORF6 cells (derived from type
293 human embryonic kidney cells), which compliment for E1 and E4
deficiencies, to produce an adenoviral Egr-1/TNF-.alpha. E1.sup.-,
E3.sup.-, E4.sup.- vector having the sequence of SEQ ID NO: 1.
EXAMPLE 2
[0060] The following example demonstrates the use of an adenoviral
vector prepared in accordance with the present invention to treat
or prevent tumors in a host.
[0061] Three treatment groups were established, each comprising
eight nude mice having radio-resistant human squameous tumor cell
line (SQ-20B) xenograft tumors. The first treatment group received
a dose of 5.times.10.sup.10 particle units (PU) of the adenoviral
particles of Example 1 (in a total volume of 32 .mu.l with a viral
buffer) by injection directly into the tumor tissue at five sites
(four injections around the periphery of each tumor and one
injection into the center of each tumor) at days 0, 4, 7, and 11.
The second treatment group received the same doses of adenoviral
vector administered in conjunction with exposure of the tumor to 5
Gy of infrared radiation on days 0-4 and 7-9 (totaling 40 Gy of
radiation). The third treatment group received only the infrared
radiation.
[0062] Significant tumor regression was observed in the first
treatment group by day four, while no regression was observed in
the second treatment group until day 11. At day 11, one animal in
the first treatment group and three animals in the second treatment
group had no visible tumor. After 62 days, all eight mice in the
second treatment group were cured (i.e., no visible tumors were
present), while six mice of the first group were cured, and only
one mouse of the third group (no vector) was cured.
[0063] The results of this example demonstrate that an adenoviral
vector according to the present invention can be successfully used
to treat tumors in a host.
EXAMPLE 3
[0064] The following example demonstrates the use of an adenoviral
vector prepared in accordance with the present invention to treat
or prevent tumors in a host.
[0065] Radio-resistant human esophageal tumor cells derived from
patient samples were injected into the right hind limb of nude mice
in a single dose of 5.times.10.sup.6 cells, and the mice were
randomly divided into four treatment groups. Group 1 was treated
only with a buffer solution, which was administered by direct
injection into the tumor on day 0 and day 3. Group 2 received
infrared radiation treatment consisting of 4 Gy doses of radiation
administered on days 0, 1, 3 and 4 (for a total IR dose of 16 Gy).
Group 3 was treated with the adenoviral vector of Example 1 (at
4.times.10.sup.8 PU), which was administered by direct injection
into the tumor on day 0 and day 3. Group 4 received the adenoviral
vector treatment and the infrared radiation treatment.
[0066] Tumor volumes and animals weights were measured over 48
days. The tumor volumes were plotted against time to yield the
tumor volume curves presented in FIG. 2, wherein the diamond-shaped
data points (.upsilon.) represent data from group 1, the circular
data points (.lambda.) represent data from group 2, triangular data
points (.sigma.) represent data from group 3, and the square data
points (.nu.) represent data from group 4. All animals remained
healthy and no untoward effects were observed throughout the study.
Animals from groups 1 and 2, however, were sacrificed at day 36 due
to the large tumor burden (greater than 10% of body weight).
[0067] The tumor volume curves demonstrate substantially less tumor
growth in animals treated with the vector and radiation (group 4)
as compared to those treated with radiation alone (group 2) or the
vector alone (group 3). This data demonstrates that the adenoviral
vector of the present invention can be successfully used to treat a
tumor in a host.
EXAMPLE 4
[0068] The following example demonstrates the use of an adenoviral
vector prepared in accordance with the present invention to treat
or prevent tumors in a host.
[0069] Radio-resistant esophageal tumor cells derived from patient
samples were injected into the right hind limb of nude mice in a
single dose of 5.times.10.sup.6 cells, and the mice were randomly
divided into four treatment groups. Group 1 was treated only with a
buffer solution, which was administered by direct injection into
the tumor on days 0, 3, 7 and 10. Group 2 received infrared
radiation treatment consisting of 3 Gy doses of radiation
administered on days 0, 1, 3, 4, 7, 8, 10 and 11 (for a total IR
dose of 24 Gy). Group 3 was treated with the adenoviral vector of
Example 1 (at 4.times.10.sup.8 PU), which was administered by
direct injection into the tumor on days 0, 3, 7 and 10. Group 4
received the adenoviral vector treatment and the infrared radiation
treatment.
[0070] Tumor volumes and animals weights were measured over 22
days. The tumor volumes were plotted against time to provide the
tumor volume curves presented in FIG. 3, wherein the diamond-shaped
data points (.upsilon.) represent data from group 1, the circular
data points (.lambda.) represent data from group 2, triangular data
points (.sigma.) represent data from group 3, and the square data
points (.nu.) represent data from group 4. All animals remained
healthy and no untoward effects were observed. Animals from groups
1 and 2, however, were sacrificed at day 15 due to the large tumor
burden (greater than 10% of body weight).
[0071] The tumor volume curves demonstrate substantially less tumor
growth in animals treated with the vector and radiation (group 4)
as compared to those treated with radiation alone (group 2) or the
vector alone (group 3). This data demonstrates that the adenoviral
vector of the present invention can be successfully used to treat a
tumor in a host.
[0072] All of the references cited herein, including patents,
patent applications, and publications, are hereby incorporated in
their entireties by reference.
[0073] While this invention has been described with an emphasis
upon preferred embodiments, it will be obvious to those of ordinary
skill in the art that variations of the preferred embodiments may
be used and that it is intended that the invention may be practiced
otherwise than as specifically described herein. Accordingly, this
invention includes all modifications encompassed within the spirit
and scope of the invention as defined by the following claims.
Sequence CWU 1
1
2 1 32798 DNA Artificial Sequence Synthetic construct 1 catcatcata
atatacctta ttttggattg aagccaatat gataatgagg gggtggagtt 60
tgtgacgtgg cgcggggcgt gggaacgggg cgggtgacgt agtagtgtgg cggaagtgtg
120 atgttgcaag tgtggcggaa cacatgtaag cgccggatgt ggtaaaagtg
acgtttttgg 180 tgtgcgccgg tgtatacggg aagtgacaat tttcgcgcgg
ttttaggcgg atgttgtagt 240 aaatttgggc gtaaccaagt aatatttggc
cattttcgcg ggaaaactga ataagaggaa 300 gtgaaatctg aataattctg
tgttactcat agcgcgtaat atttgtctag ggccgcgggg 360 actttgaccg
tttacgtgga gactcgccca ggtgtttttc tcaggtgttt tccgcgttcc 420
gggtcaaagt tggcgtttta ttattatagt cagctctaga ctagatgcgc cgacccggaa
480 acgccatata aggagcagga aggatccccc gccggaacag accttatttg
ggcagcgcct 540 tatatggagt ggcccaatat ggccctgccg cttccggctc
tgggaggagg ggcgagcggg 600 ggttggggcg ggggcaagct gggaactcca
ggcgcctggc ccgggaggcc actgctgctg 660 ttccaatact aggctttcca
ggagcctgag cgctcgcgat gccggagcgg gtcgcagggt 720 ggaggtgccc
accactcttg gatgggaggg cttcacgtca ctccgggtcc tcccggccgg 780
tccttccata ttagggcttc ctgcttccca tatatggcca tgtacgtcac ggcggaggcg
840 ggcccgtgct gttccagacc cttgaaatag aggccgattc ggggagtcgc
gagagatccc 900 agcgcgcaga acttggggag ccgccgccgc gattcgccgc
cgccgccagc ttccggtcga 960 ggaactgaaa aaccagaaag ttaactgggt
aagtttagtc tttttgtctt ttatttcagg 1020 tcccggatcc ggtggtggtg
caaatcaaag aactgctcct cagtggatgt tgcctttact 1080 tctaggcctg
tacggaagtg ttacttctgc tctaaaagct gcggaattgt acccgcggcc 1140
gcaaagggaa caaaagctgg gtaccgagct cgaatggggg gggggggggg gtactgaccc
1200 acggctccac cctctctccc ctggaaagga caccatgagc actgaaagca
tgatccggga 1260 cgtggagctg gccgaggagg cgctccccaa gaagacaggg
gggccccagg gctccaggcg 1320 gtgcttgttc ctcagcctct tctccttcct
gatcgtggca ggcgccacca cgctcttctg 1380 cctgctgcac tttggagtga
tcggccccca gagggaagag tcccccaggg acctctctct 1440 aatcagccct
ctggcccagg cagtcagatc atcttctcga accccgagtg acaagcctgt 1500
agcccatgtt gtagcaaacc ctcaagctga ggggcagctc cagtggctga accgccgggc
1560 caatgccctc ctggccaatg gcgtggagct gagagataac cagctggtgg
tgccatcaga 1620 gggcctgtac ctcatctact cccaggtcct cttcaagggc
caaggctgcc cctccaccca 1680 tgtgctcctc acccacacca tcagccgcat
cgccgtctcc taccagacca aggtcaacct 1740 cctctctgcc atcaagagcc
cctgccagag ggagacccca gagggggctg aggccaagcc 1800 ctggtatgag
cccatctatc tgggaggggt cttccagctg gagaagggtg accgactcag 1860
cgctgagatc aatcggcccg actatctcga ctttgccgag tctgggcagg tctactttgg
1920 gatcattgcc ctgtgaggag gacgaacatc caaccttccc aaacgcctcc
cctgccccaa 1980 tccctttatt accccctcct tcagacaccc tcaacctctt
ctggctcaaa aagagaattg 2040 ggggcttagg gtcggaaccc aagcttgata
tcgaattcct gcagcccggg ggatccacta 2100 gttctagagc ggccgcgggg
atccagacat gataagatac attgatgagt ttggacaaac 2160 cacaactaga
atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt 2220
atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat
2280 gtttcaggtt cagggggagg tgtgggaggt tttttcggat cctctagagt
cgactagagt 2340 ggaaggtgct gaggtacgat gagacccgca ccaggtgcag
accctgcgag tgtggcggta 2400 aacatattag gaaccagcct gtgatgctgg
atgtgaccga ggagctgagg cccgatcact 2460 tggtgctggc ctgcacccgc
gctgagtttg gctctagcga tgaagataca gattgaggta 2520 ctgaaatgtg
tgggcgtggc ttaagggtgg gaaagaatat ataaggtggg ggtcttatgt 2580
agttttgtat ctgttttgca gcagccgccg ccgccatgag caccaactcg tttgatggaa
2640 gcattgtgag ctcatatttg acaacgcgca tgcccccatg ggccggggtg
cgtcagaatg 2700 tgatgggctc cagcattgat ggtcgccccg tcctgcccgc
aaactctact accttgacct 2760 acgagaccgt gtctggaacg ccgttggaga
ctgcagcctc cgccgccgct tcagccgctg 2820 cagccaccgc ccgcgggatt
gtgactgact ttgctttcct gagcccgctt gcaagcagtg 2880 cagcttcccg
ttcatccgcc cgcgatgaca agttgacggc tcttttggca caattggatt 2940
ctttgacccg ggaacttaat gtcgtttctc agcagctgtt ggatctgcgc cagcaggttt
3000 ctgccctgaa ggcttcctcc cctcccaatg cggtttaaaa cataaataaa
aaaccagact 3060 ctgtttggat ttggatcaag caagtgtctt gctgtcttta
tttaggggtt ttgcgcgcgc 3120 ggtaggcccg ggaccagcgg tctcggtcgt
tgagggtcct gtgtattttt tccaggacgt 3180 ggtaaaggtg actctggatg
ttcagataca tgggcataag cccgtctctg gggtggaggt 3240 agcaccactg
cagagcttca tgctgcgggg tggtgttgta gatgatccag tcgtagcagg 3300
agcgctgggc gtggtgccta aaaatgtctt tcagtagcaa gctgattgcc aggggcaggc
3360 ccttggtgta agtgtttaca aagcggttaa gctgggatgg gtgcatacgt
ggggatatga 3420 gatgcatctt ggactgtatt tttaggttgg ctatgttccc
agccatatcc ctccggggat 3480 tcatgttgtg cagaaccacc agcacagtgt
atccggtgca cttgggaaat ttgtcatgta 3540 gcttagaagg aaatgcgtgg
aagaacttgg agacgccctt gtgacctcca agattttcca 3600 tgcattcgtc
cataatgatg gcaatgggcc cacgggcggc ggcctgggcg aagatatttc 3660
tgggatcact aacgtcatag ttgtgttcca ggatgagatc gtcataggcc atttttacaa
3720 agcgcgggcg gagggtgcca gactgcggta taatggttcc atccggccca
ggggcgtagt 3780 taccctcaca gatttgcatt tcccacgctt tgagttcaga
tggggggatc atgtctacct 3840 gcggggcgat gaagaaaacg gtttccgggg
taggggagat cagctgggaa gaaagcaggt 3900 tcctgagcag ctgcgactta
ccgcagccgg tgggcccgta aatcacacct attaccgggt 3960 gcaactggta
gttaagagag ctgcagctgc cgtcatccct gagcaggggg gccacttcgt 4020
taagcatgtc cctgactcgc atgttttccc tgaccaaatc cgccagaagg cgctcgccgc
4080 ccagcgatag cagttcttgc aaggaagcaa agtttttcaa cggtttgaga
ccgtccgccg 4140 taggcatgct tttgagcgtt tgaccaagca gttccaggcg
gtcccacagc tcggtcacct 4200 gctctacggc atctcgatcc agcatatctc
ctcgtttcgc gggttggggc ggctttcgct 4260 gtacggcagt agtcggtgct
cgtccagacg ggccagggtc atgtctttcc acgggcgcag 4320 ggtcctcgtc
agcgtagtct gggtcacggt gaaggggtgc gctccgggct gcgcgctggc 4380
cagggtgcgc ttgaggctgg tcctgctggt gctgaagcgc tgccggtctt cgccctgcgc
4440 gtcggccagg tagcatttga ccatggtgtc atagtccagc ccctccgcgg
cgtggccctt 4500 ggcgcgcagc ttgcccttgg aggaggcgcc gcacgagggg
cagtgcagac ttttgagggc 4560 gtagagcttg ggcgcgagaa ataccgattc
cggggagtag gcatccgcgc cgcaggcccc 4620 gcagacggtc tcgcattcca
cgagccaggt gagctctggc cgttcggggt caaaaaccag 4680 gtttccccca
tgctttttga tgcgtttctt acctctggtt tccatgagcc ggtgtccacg 4740
ctcggtgacg aaaaggctgt ccgtgtcccc gtatacagac ttgagaggcc tgtcctcgag
4800 cggtgttccg cggtcctcct cgtatagaaa ctcggaccac tctgagacaa
aggctcgcgt 4860 ccaggccagc acgaaggagg ctaagtggga ggggtagcgg
tcgttgtcca ctagggggtc 4920 cactcgctcc agggtgtgaa gacacatgtc
gccctcttcg gcatcaagga aggtgattgg 4980 tttgtaggtg taggccacgt
gaccgggtgt tcctgaaggg gggctataaa agggggtggg 5040 ggcgcgttcg
tcctcactct cttccgcatc gctgtctgcg agggccagct gttggggtga 5100
gtactccctc tgaaaagcgg gcatgacttc tgcgctaaga ttgtcagttt ccaaaaacga
5160 ggaggatttg atattcacct ggcccgcggt gatgcctttg agggtggccg
catccatctg 5220 gtcagaaaag acaatctttt tgttgtcaag cttggtggca
aacgacccgt agagggcgtt 5280 ggacagcaac ttggcgatgg agcgcagggt
ttggtttttg tcgcgatcgg cgcgctcctt 5340 ggccgcgatg tttagctgca
cgtattcgcg cgcaacgcac cgccattcgg gaaagacggt 5400 ggtgcgctcg
tcgggcacca ggtgcacgcg ccaaccgcgg ttgtgcaggg tgacaaggtc 5460
aacgctggtg gctacctctc cgcgtaggcg ctcgttggtc cagcagaggc ggccgccctt
5520 gcgcgagcag aatggcggta gggggtctag ctgcgtctcg tccggggggt
ctgcgtccac 5580 ggtaaagacc ccgggcagca ggcgcgcgtc gaagtagtct
atcttgcatc cttgcaagtc 5640 tagcgcctgc tgccatgcgc gggcggcaag
cgcgcgctcg tatgggttga gtgggggacc 5700 ccatggcatg gggtgggtga
gcgcggaggc gtacatgccg caaatgtcgt aaacgtagag 5760 gggctctctg
agtattccaa gatatgtagg gtagcatctt ccaccgcgga tgctggcgcg 5820
cacgtaatcg tatagttcgt gcgagggagc gaggaggtcg ggaccgaggt tgctacgggc
5880 gggctgctct gctcggaaga ctatctgcct gaagatggca tgtgagttgg
atgatatggt 5940 tggacgctgg aagacgttga agctggcgtc tgtgagacct
accgcgtcac gcacgaagga 6000 ggcgtaggag tcgcgcagct tgttgaccag
ctcggcggtg acctgcacgt ctagggcgca 6060 gtagtccagg gtttccttga
tgatgtcata cttatcctgt cccttttttt tccacagctc 6120 gcggttgagg
acaaactctt cgcggtcttt ccagtactct tggatcggaa acccgtcggc 6180
ctccgaacgg taagagccta gcatgtagaa ctggttgacg gcctggtagg cgcagcatcc
6240 cttttctacg ggtagcgcgt atgcctgcgc ggccttccgg agcgaggtgt
gggtgagcgc 6300 aaaggtgtcc ctgaccatga ctttgaggta ctggtatttg
aagtcagtgt cgtcgcatcc 6360 gccctgctcc cagagcaaaa agtccgtgcg
ctttttggaa cgcggatttg gcagggcgaa 6420 ggtgacatcg ttgaagagta
tctttcccgc gcgaggcata aagttgcgtg tgatgcggaa 6480 gggtcccggc
acctcggaac ggttgttaat tacctgggcg gcgagcacga tctcgtcaaa 6540
gccgttgatg ttgtggccca caatgtaaag ttccaagaag cgcgggatgc ccttgatgga
6600 aggcaatttt ttaagttcct cgtaggtgag ctcttcaggg gagctgagcc
cgtgctctga 6660 aagggcccag tctgcaagat gagggttgga agcgacgaat
gagctccaca ggtcacgggc 6720 cattagcatt tgcaggtggt cgcgaaaggt
cctaaactgg cgacctatgg ccattttttc 6780 tggggtgatg cagtagaagg
taagcgggtc ttgttcccag cggtcccatc caaggttcgc 6840 ggctaggtct
cgcgcggcag tcactagagg ctcatctccg ccgaacttca tgaccagcat 6900
gaagggcacg agctgcttcc caaaggcccc catccaagta taggtctcta catcgtaggt
6960 gacaaagaga cgctcggtgc gaggatgcga gccgatcggg aagaactgga
tctcccgcca 7020 ccaattggag gagtggctat tgatgtggtg aaagtagaag
tccctgcgac gggccgaaca 7080 ctcgtgctgg cttttgtaaa aacgtgcgca
gtactggcag cggtgcacgg gctgtacatc 7140 ctgcacgagg ttgacctgac
gaccgcgcac aaggaagcag agtgggaatt tgagcccctc 7200 gcctggcggg
tttggctggt ggtcttctac ttcggctgct tgtccttgac cgtctggctg 7260
ctcgagggga gttacggtgg atcggaccac cacgccgcgc gagcccaaag tccagatgtc
7320 cgcgcgcggc ggtcggagct tgatgacaac atcgcgcaga tgggagctgt
ccatggtctg 7380 gagctcccgc ggcgtcaggt caggcgggag ctcctgcagg
tttacctcgc atagacgggt 7440 cagggcgcgg gctagatcca ggtgatacct
aatttccagg ggctggttgg tggcggcgtc 7500 gatggcttgc aagaggccgc
atccccgcgg cgcgactacg gtaccgcgcg gcgggcggtg 7560 ggccgcgggg
gtgtccttgg atgatgcatc taaaagcggt gacgcgggcg agcccccgga 7620
ggtagggggg gctccggacc cgccgggaga gggggcaggg gcacgtcggc gccgcgcgcg
7680 ggcaggagct ggtgctgcgc gcgtaggttg ctggcgaacg cgacgacgcg
gcggttgatc 7740 tcctgaatct ggcgcctctg cgtgaagacg acgggcccgg
tgagcttgag cctgaaagag 7800 agttcgacag aatcaatttc ggtgtcgttg
acggcggcct ggcgcaaaat ctcctgcacg 7860 tctcctgagt tgtcttgata
ggcgatctcg gccatgaact gctcgatctc ttcctcctgg 7920 agatctccgc
gtccggctcg ctccacggtg gcggcgaggt cgttggaaat gcgggccatg 7980
agctgcgaga aggcgttgag gcctccctcg ttccagacgc ggctgtagac cacgccccct
8040 tcggcatcgc gggcgcgcat gaccacctgc gcgagattga gctccacgtg
ccgggcgaag 8100 acggcgtagt ttcgcaggcg ctgaaagagg tagttgaggg
tggtggcggt gtgttctgcc 8160 acgaagaagt acataaccca gcgtcgcaac
gtggattcgt tgatatcccc caaggcctca 8220 aggcgctcca tggcctcgta
gaagtccacg gcgaagttga aaaactggga gttgcgcgcc 8280 gacacggtta
actcctcctc cagaagacgg atgagctcgg cgacagtgtc gcgcacctcg 8340
cgctcaaagg ctacaggggc ctcttcttct tcttcaatct cctcttccat aagggcctcc
8400 ccttcttctt cttctggcgg cggtggggga ggggggacac ggcggcgacg
acggcgcacc 8460 gggaggcggt cgacaaagcg ctcgatcatc tccccgcggc
gacggcgcat ggtctcggtg 8520 acggcgcggc cgttctcgcg ggggcgcagt
tggaagacgc cgcccgtcat gtcccggtta 8580 tgggttggcg gggggctgcc
atgcggcagg gatacggcgc taacgatgca tctcaacaat 8640 tgttgtgtag
gtactccgcc gccgagggac ctgagcgagt ccgcatcgac cggatcggaa 8700
aacctctcga gaaaggcgtc taaccagtca cagtcgcaag gtaggctgag caccgtggcg
8760 ggcggcagcg ggcggcggtc ggggttgttt ctggcggagg tgctgctgat
gatgtaatta 8820 aagtaggcgg tcttgagacg gcggatggtc gacagaagca
ccatgtcctt gggtccggcc 8880 tgctgaatgc gcaggcggtc ggccatgccc
caggcttcgt tttgacatcg gcgcaggtct 8940 ttgtagtagt cttgcatgag
cctttctacc ggcacttctt cttctccttc ctcttgtcct 9000 gcatctcttg
catctatcgc tgcggcggcg gcggagtttg gccgtaggtg gcgccctctt 9060
cctcccatgc gtgtgacccc gaagcccctc atcggctgaa gcagggctag gtcggcgaca
9120 acgcgctcgg ctaatatggc ctgctgcacc tgcgtgaggg tagactggaa
gtcatccatg 9180 tccacaaagc ggtggtatgc gcccgtgttg atggtgtaag
tgcagttggc cataacggac 9240 cagttaacgg tctggtgacc cggctgcgag
agctcggtgt acctgagacg cgagtaagcc 9300 ctcgagtcaa atacgtagtc
gttgcaagtc cgcaccaggt actggtatcc caccaaaaag 9360 tgcggcggcg
gctggcggta gaggggccag cgtagggtgg ccggggctcc gggggcgaga 9420
tcttccaaca taaggcgatg atatccgtag atgtacctgg acatccaggt gatgccggcg
9480 gcggtggtgg aggcgcgcgg aaagtcgcgg acgcggttcc agatgttgcg
cagcggcaaa 9540 aagtgctcca tggtcgggac gctctggccg gtcaggcgcg
cgcaatcgtt gacgctctag 9600 ccgtgcaaaa ggagagcctg taagcgggca
ctcttccgtg gtctggtgga taaattcgca 9660 agggtatcat ggcggacgac
cggggttcga gccccgtatc cggccgtccg ccgtgatcca 9720 tgcggttacc
gcccgcgtgt cgaacccagg tgtgcgacgt cagacaacgg gggagtgctc 9780
cttttggctt ccttccaggc gcggcggctg ctgcgctagc ttttttggcc actggccgcg
9840 cgcagcgtaa gcggttaggc tggaaagcga aagcattaag tggctcgctc
cctgtagccg 9900 gagggttatt ttccaagggt tgagtcgcgg gacccccggt
tcgagtctcg gaccggccgg 9960 actgcggcga acgggggttt gcctccccgt
catgcaagac cccgcttgca aattcctccg 10020 gaaacaggga cgagcccctt
ttttgctttt cccagatgca tccggtgctg cggcagatgc 10080 gcccccctcc
tcagcagcgg caagagcaag agcagcggca gacatgcagg gcaccctccc 10140
ctcctcctac cgcgtcagga ggggcgacat ccgcggttga cgcggcagca gatggtgatt
10200 acgaaccccc gcggcgccgg gcccggcact acctggactt ggaggagggc
gagggcctgg 10260 cgcggctagg agcgccctct cctgagcggt acccaagggt
gcagctgaag cgtgatacgc 10320 gtgaggcgta cgtgccgcgg cagaacctgt
ttcgcgaccg cgagggagag gagcccgagg 10380 agatgcggga tcgaaagttc
cacgcagggc gcgagctgcg gcatggcctg aatcgcgagc 10440 ggttgctgcg
cgaggaggac tttgagcccg acgcgcgaac cgggattagt cccgcgcgcg 10500
cacacgtggc ggccgccgac ctggtaaccg catacgagca gacggtgaac caggagatta
10560 actttcaaaa aagctttaac aaccacgtgc gtacgcttgt ggcgcgcgag
gaggtggcta 10620 taggactgat gcatctgtgg gactttgtaa gcgcgctgga
gcaaaaccca aatagcaagc 10680 cgctcatggc gcagctgttc cttatagtgc
agcacagcag ggacaacgag gcattcaggg 10740 atgcgctgct aaacatagta
gagcccgagg gccgctggct gctcgatttg ataaacatcc 10800 tgcagagcat
agtggtgcag gagcgcagct tgagcctggc tgacaaggtg gccgccatca 10860
actattccat gcttagcctg ggcaagtttt acgcccgcaa gatataccat accccttacg
10920 ttcccataga caaggaggta aagatcgagg ggttctacat gcgcatggcg
ctgaaggtgc 10980 ttaccttgag cgacgacctg ggcgtttatc gcaacgagcg
catccacaag gccgtgagcg 11040 tgagccggcg gcgcgagctc agcgaccgcg
agctgatgca cagcctgcaa agggccctgg 11100 ctggcacggg cagcggcgat
agagaggccg agtcctactt tgacgcgggc gctgacctgc 11160 gctgggcccc
aagccgacgc gccctggagg cagctggggc cggacctggg ctggcggtgg 11220
cacccgcgcg cgctggcaac gtcggcggcg tggaggaata tgacgaggac gatgagtacg
11280 agccagagga cggcgagtac taagcggtga tgtttctgat cagatgatgc
aagacgcaac 11340 ggacccggcg gtgcgggcgg cgctgcagag ccagccgtcc
ggccttaact ccacggacga 11400 ctggcgccag gtcatggacc gcatcatgtc
gctgactgcg cgcaatcctg acgcgttccg 11460 gcagcagccg caggccaacc
ggctctccgc aattctggaa gcggtggtcc cggcgcgcgc 11520 aaaccccacg
cacgagaagg tgctggcgat cgtaaacgcg ctggccgaaa acagggccat 11580
ccggcccgac gaggccggcc tggtctacga cgcgctgctt cagcgcgtgg ctcgttacaa
11640 cagcggcaac gtgcagacca acctggaccg gctggtgggg gatgtgcgcg
aggccgtggc 11700 gcagcgtgag cgcgcgcagc agcagggcaa cctgggctcc
atggttgcac taaacgcctt 11760 cctgagtaca cagcccgcca acgtgccgcg
gggacaggag gactacacca actttgtgag 11820 cgcactgcgg ctaatggtga
ctgagacacc gcaaagtgag gtgtaccagt ctgggccaga 11880 ctattttttc
cagaccagta gacaaggcct gcagaccgta aacctgagcc aggctttcaa 11940
aaacttgcag gggctgtggg gggtgcgggc tcccacaggc gaccgcgcga ccgtgtctag
12000 cttgctgacg cccaactcgc gcctgttgct gctgctaata gcgcccttca
cggacagtgg 12060 cagcgtgtcc cgggacacat acctaggtca cttgctgaca
ctgtaccgcg aggccatagg 12120 tcaggcgcat gtggacgagc atactttcca
ggagattaca agtgtcagcc gcgcgctggg 12180 gcaggaggac acgggcagcc
tggaggcaac cctaaactac ctgctgacca accggcggca 12240 gaagatcccc
tcgttgcaca gtttaaacag cgaggaggag cgcattttgc gctacgtgca 12300
gcagagcgtg agccttaacc tgatgcgcga cggggtaacg cccagcgtgg cgctggacat
12360 gaccgcgcgc aacatggaac cgggcatgta tgcctcaaac cggccgttta
tcaaccgcct 12420 aatggactac ttgcatcgcg cggccgccgt gaaccccgag
tatttcacca atgccatctt 12480 gaacccgcac tggctaccgc cccctggttt
ctacaccggg ggattcgagg tgcccgaggg 12540 taacgatgga ttcctctggg
acgacataga cgacagcgtg ttttccccgc aaccgcagac 12600 cctgctagag
ttgcaacagc gcgagcaggc agaggcggcg ctgcgaaagg aaagcttccg 12660
caggccaagc agcttgtccg atctaggcgc tgcggccccg cggtcagatg ctagtagccc
12720 atttccaagc ttgatagggt ctcttaccag cactcgcacc acccgcccgc
gcctgctggg 12780 cgaggaggag tacctaaaca actcgctgct gcagccgcag
cgcgaaaaaa acctgcctcc 12840 ggcatttccc aacaacggga tagagagcct
agtggacaag atgagtagat ggaagacgta 12900 cgcgcaggag cacagggacg
tgccaggccc gcgcccgccc acccgtcgtc aaaggcacga 12960 ccgtcagcgg
ggtctggtgt gggaggacga tgactcggca gacgacagca gcgtcctgga 13020
tttgggaggg agtggcaacc cgtttgcgca ccttcgcccc aggctgggga gaatgtttta
13080 aaaaaaaaaa agcatgatgc aaaataaaaa actcaccaag gccatggcac
cgagcgttgg 13140 ttttcttgta ttccccttag tatgcggcgc gcggcgatgt
atgaggaagg tcctcctccc 13200 tcctacgaga gtgtggtgag cgcggcgcca
gtggcggcgg cgctgggttc tcccttcgat 13260 gctcccctgg acccgccgtt
tgtgcctccg cggtacctgc ggcctaccgg ggggagaaac 13320 agcatccgtt
actctgagtt ggcaccccta ttcgacacca cccgtgtgta cctggtggac 13380
aacaagtcaa cggatgtggc atccctgaac taccagaacg accacagcaa ctttctgacc
13440 acggtcattc aaaacaatga ctacagcccg ggggaggcaa gcacacagac
catcaatctt 13500 gacgaccggt cgcactgggg cggcgacctg aaaaccatcc
tgcataccaa catgccaaat 13560 gtgaacgagt tcatgtttac caataagttt
aaggcgcggg tgatggtgtc gcgcttgcct 13620 actaaggaca atcaggtgga
gctgaaatac gagtgggtgg agttcacgct gcccgagggc 13680 aactactccg
agaccatgac catagacctt atgaacaacg cgatcgtgga gcactacttg 13740
aaagtgggca gacagaacgg ggttctggaa agcgacatcg gggtaaagtt tgacacccgc
13800 aacttcagac tggggtttga ccccgtcact ggtcttgtca tgcctggggt
atatacaaac 13860 gaagccttcc atccagacat cattttgctg ccaggatgcg
gggtggactt cacccacagc 13920 cgcctgagca acttgttggg catccgcaag
cggcaaccct tccaggaggg ctttaggatc 13980 acctacgatg atctggaggg
tggtaacatt cccgcactgt tggatgtgga cgcctaccag 14040 gcgagcttga
aagatgacac cgaacagggc gggggtggcg caggcggcag caacagcagt 14100
ggcagcggcg cggaagagaa ctccaacgcg gcagccgcgg caatgcagcc ggtggaggac
14160 atgaacgatc atgccattcg cggcgacacc tttgccacac gggctgagga
gaagcgcgct 14220 gaggccgaag cagcggccga agctgccgcc cccgctgcgc
aacccgaggt cgagaagcct 14280 cagaagaaac cggtgatcaa acccctgaca
gaggacagca agaaacgcag ttacaaccta 14340 ataagcaatg acagcacctt
cacccagtac cgcagctggt accttgcata caactacggc 14400 gaccctcaga
ccggaatccg ctcatggacc ctgctttgca ctcctgacgt aacctgcggc 14460
tcggagcagg tctactggtc gttgccagac atgatgcaag accccgtgac cttccgctcc
14520 acgcgccaga tcagcaactt tccggtggtg ggcgccgagc tgttgcccgt
gcactccaag 14580 agcttctaca acgaccaggc cgtctactcc caactcatcc
gccagtttac ctctctgacc 14640 cacgtgttca atcgctttcc cgagaaccag
attttggcgc gcccgccagc ccccaccatc 14700 accaccgtca gtgaaaacgt
tcctgctctc acagatcacg ggacgctacc gctgcgcaac 14760 agcatcggag
gagtccagcg agtgaccatt actgacgcca gacgccgcac ctgcccctac 14820
gtttacaagg ccctgggcat agtctcgccg cgcgtcctat cgagccgcac tttttgagca
14880 agcatgtcca tccttatatc gcccagcaat aacacaggct ggggcctgcg
cttcccaagc 14940 aagatgtttg gcggggccaa gaagcgctcc gaccaacacc
cagtgcgcgt gcgcgggcac 15000 taccgcgcgc
cctggggcgc gcacaaacgc ggccgcactg ggcgcaccac cgtcgatgac 15060
gccatcgacg cggtggtgga ggaggcgcgc aactacacgc ccacgccgcc accagtgtcc
15120 acagtggacg cggccattca gaccgtggtg cgcggagccc ggcgctatgc
taaaatgaag 15180 agacggcgga ggcgcgtagc acgtcgccac cgccgccgac
ccggcactgc cgcccaacgc 15240 gcggcggcgg ccctgcttaa ccgcgcacgt
cgcaccggcc gacgggcggc catgcgggcc 15300 gctcgaaggc tggccgcggg
tattgtcact gtgcccccca ggtccaggcg acgagcggcc 15360 gccgcagcag
ccgcggccat tagtgctatg actcagggtc gcaggggcaa cgtgtattgg 15420
gtgcgcgact cggttagcgg cctgcgcgtg cccgtgcgca cccgcccccc gcgcaactag
15480 attgcaagaa aaaactactt agactcgtac tgttgtatgt atccagcggc
ggcggcgcgc 15540 aacgaagcta tgtccaagcg caaaatcaaa gaagagatgc
tccaggtcat cgcgccggag 15600 atctatggcc ccccgaagaa ggaagagcag
gattacaagc cccgaaagct aaagcgggtc 15660 aaaaagaaaa agaaagatga
tgatgatgaa cttgacgacg aggtggaact gctgcacgct 15720 accgcgccca
ggcgacgggt acagtggaaa ggtcgacgcg taaaacgtgt tttgcgaccc 15780
ggcaccaccg tagtctttac gcccggtgag cgctccaccc gcacctacaa gcgcgtgtat
15840 gatgaggtgt acggcgacga ggacctgctt gagcaggcca acgagcgcct
cggggagttt 15900 gcctacggaa agcggcataa ggacatgctg gcgttgccgc
tggacgaggg caacccaaca 15960 cctagcctaa agcccgtaac actgcagcag
gtgctgcccg cgcttgcacc gtccgaagaa 16020 aagcgcggcc taaagcgcga
gtctggtgac ttggcaccca ccgtgcagct gatggtaccc 16080 aagcgccagc
gactggaaga tgtcttggaa aaaatgaccg tggaacctgg gctggagccc 16140
gaggtccgcg tgcggccaat caagcaggtg gcgccgggac tgggcgtgca gaccgtggac
16200 gttcagatac ccactaccag tagcaccagt attgccaccg ccacagaggg
catggagaca 16260 caaacgtccc cggttgcctc agcggtggcg gatgccgcgg
tgcaggcggt cgctgcggcc 16320 gcgtccaaga cctctacgga ggtgcaaacg
gacccgtgga tgtttcgcgt ttcagccccc 16380 cggcgcccgc gcggttcgag
gaagtacggc gccgccagcg cgctactgcc cgaatatgcc 16440 ctacatcctt
ccattgcgcc tacccccggc tatcgtggct acacctaccg ccccagaaga 16500
cgagcaacta cccgacgccg aaccaccact ggaacccgcc gccgccgtcg ccgtcgccag
16560 cccgtgctgg ccccgatttc cgtgcgcagg gtggctcgcg aaggaggcag
gaccctggtg 16620 ctgccaacag cgcgctacca ccccagcatc gtttaaaagc
cggtctttgt ggttcttgca 16680 gatatggccc tcacctgccg cctccgtttc
ccggtgccgg gattccgagg aagaatgcac 16740 cgtaggaggg gcatggccgg
ccacggcctg acgggcggca tgcgtcgtgc gcaccaccgg 16800 cggcggcgcg
cgtcgcaccg tcgcatgcgc ggcggtatcc tgcccctcct tattccactg 16860
atcgccgcgg cgattggcgc cgtgcccgga attgcatccg tggccttgca ggcgcagaga
16920 cactgattaa aaacaagttg catgtggaaa aatcaaaata aaaagtctgg
actctcacgc 16980 tcgcttggtc ctgtaactat tttgtagaat ggaagacatc
aactttgcgt ctctggcccc 17040 gcgacacggc tcgcgcccgt tcatgggaaa
ctggcaagat atcggcacca gcaatatgag 17100 cggtggcgcc ttcagctggg
gctcgctgtg gagcggcatt aaaaatttcg gttccaccgt 17160 taagaactat
ggcagcaagg cctggaacag cagcacaggc cagatgctga gggataagtt 17220
gaaagagcaa aatttccaac aaaaggtggt agatggcctg gcctctggca ttagcggggt
17280 ggtggacctg gccaaccagg cagtgcaaaa taagattaac agtaagcttg
atccccgccc 17340 tcccgtagag gagcctccac cggccgtgga gacagtgtct
ccagaggggc gtggcgaaaa 17400 gcgtccgcgc cccgacaggg aagaaactct
ggtgacgcaa atagacgagc ctccctcgta 17460 cgaggaggca ctaaagcaag
gcctgcccac cacccgtccc atcgcgccca tggctaccgg 17520 agtgctgggc
cagcacacac ccgtaacgct ggacctgcct ccccccgccg acacccagca 17580
gaaacctgtg ctgccaggcc cgaccgccgt tgttgtaacc cgtcctagcc gcgcgtccct
17640 gcgccgcgcc gccagcggtc cgcgatcgtt gcggcccgta gccagtggca
actggcaaag 17700 cacactgaac agcatcgtgg gtctgggggt gcaatccctg
aagcgccgac gatgcttctg 17760 aatagctaac gtgtcgtatg tgtgtcatgt
atgcgtccat gtcgccgcca gaggagctgc 17820 tgagccgccg cgcgcccgct
ttccaagatg gctacccctt cgatgatgcc gcagtggtct 17880 tacatgcaca
tctcgggcca ggacgcctcg gagtacctga gccccgggct ggtgcagttt 17940
gcccgcgcca ccgagacgta cttcagcctg aataacaagt ttagaaaccc cacggtggcg
18000 cctacgcacg acgtgaccac agaccggtcc cagcgtttga cgctgcggtt
catccctgtg 18060 gaccgtgagg atactgcgta ctcgtacaag gcgcggttca
ccctagctgt gggtgataac 18120 cgtgtgctgg acatggcttc cacgtacttt
gacatccgcg gcgtgctgga caggggccct 18180 acttttaagc cctactctgg
cactgcctac aacgccctgg ctcccaaggg tgccccaaat 18240 ccttgcgaat
gggatgaagc tgctactgct cttgaaataa acctagaaga agaggacgat 18300
gacaacgaag acgaagtaga cgagcaagct gagcagcaaa aaactcacgt atttgggcag
18360 gcgccttatt ctggtataaa tattacaaag gagggtattc aaataggtgt
cgaaggtcaa 18420 acacctaaat atgccgataa aacatttcaa cctgaacctc
aaataggaga atctcagtgg 18480 tacgaaactg aaattaatca tgcagctggg
agagtcctta aaaagactac cccaatgaaa 18540 ccatgttacg gttcatatgc
aaaacccaca aatgaaaatg gagggcaagg cattcttgta 18600 aagcaacaaa
atggaaagct agaaagtcaa gtggaaatgc aatttttctc aactactgag 18660
gcgaccgcag gcaatggtga taacttgact cctaaagtgg tattgtacag tgaagatgta
18720 gatatagaaa ccccagacac tcatatttct tacatgccca ctattaagga
aggtaactca 18780 cgagaactaa tgggccaaca atctatgccc aacaggccta
attacattgc ttttagggac 18840 aattttattg gtctaatgta ttacaacagc
acgggtaata tgggtgttct ggcgggccaa 18900 gcatcgcagt tgaatgctgt
tgtagatttg caagacagaa acacagagct ttcataccag 18960 cttttgcttg
attccattgg tgatagaacc aggtactttt ctatgtggaa tcaggctgtt 19020
gacagctatg atccagatgt tagaattatt gaaaatcatg gaactgaaga tgaacttcca
19080 aattactgct ttccactggg aggtgtgatt aatacagaga ctcttaccaa
ggtaaaacct 19140 aaaacaggtc aggaaaatgg atgggaaaaa gatgctacag
aattttcaga taaaaatgaa 19200 ataagagttg gaaataattt tgccatggaa
atcaatctaa atgccaacct gtggagaaat 19260 ttcctgtact ccaacatagc
gctgtatttg cccgacaagc taaagtacag tccttccaac 19320 gtaaaaattt
ctgataaccc aaacacctac gactacatga acaagcgagt ggtggctccc 19380
gggttagtgg actgctacat taaccttgga gcacgctggt cccttgacta tatggacaac
19440 gtcaacccat ttaaccacca ccgcaatgct ggcctgcgct accgctcaat
gttgctgggc 19500 aatggtcgct atgtgccctt ccacatccag gtgcctcaga
agttctttgc cattaaaaac 19560 ctccttctcc tgccgggctc atacacctac
gagtggaact tcaggaagga tgttaacatg 19620 gttctgcaga gctccctagg
aaatgaccta agggttgacg gagccagcat taagtttgat 19680 agcatttgcc
tttacgccac cttcttcccc atggcccaca acaccgcctc cacgcttgag 19740
gccatgctta gaaacgacac caacgaccag tcctttaacg actatctctc cgccgccaac
19800 atgctctacc ctatacccgc caacgctacc aacgtgccca tatccatccc
ctcccgcaac 19860 tgggcggctt tccgcggctg ggccttcacg cgccttaaga
ctaaggaaac cccatcactg 19920 ggctcgggct acgaccctta ttacacctac
tctggctcta taccctacct agatggaacc 19980 ttttacctca accacacctt
taagaaggtg gccattacct ttgactcttc tgtcagctgg 20040 cctggcaatg
accgcctgct tacccccaac gagtttgaaa ttaagcgctc agttgacggg 20100
gagggttaca acgttgccca gtgtaacatg accaaagact ggttcctggt acaaatgcta
20160 gctaactaca acattggcta ccagggcttc tatatcccag agagctacaa
ggaccgcatg 20220 tactccttct ttagaaactt ccagcccatg agccgtcagg
tggtggatga tactaaatac 20280 aaggactacc aacaggtggg catcctacac
caacacaaca actctggatt tgttggctac 20340 cttgccccca ccatgcgcga
aggacaggcc taccctgcta acttccccta tccgcttata 20400 ggcaagaccg
cagttgacag cattacccag aaaaagtttc tttgcgatcg caccctttgg 20460
cgcatcccat tctccagtaa ctttatgtcc atgggcgcac tcacagacct gggccaaaac
20520 cttctctacg ccaactccgc ccacgcgcta gacatgactt ttgaggtgga
tcccatggac 20580 gagcccaccc ttctttatgt tttgtttgaa gtctttgacg
tggtccgtgt gcaccggccg 20640 caccgcggcg tcatcgaaac cgtgtacctg
cgcacgccct tctcggccgg caacgccaca 20700 acataaagaa gcaagcaaca
tcaacaacag ctgccgccat gggctccagt gagcaggaac 20760 tgaaagccat
tgtcaaagat cttggttgtg ggccatattt tttgggcacc tatgacaagc 20820
gctttccagg ctttgtttct ccacacaagc tcgcctgcgc catagtcaat acggccggtc
20880 gcgagactgg gggcgtacac tggatggcct ttgcctggaa cccgcactca
aaaacatgct 20940 acctctttga gccctttggc ttttctgacc agcgactcaa
gcaggtttac cagtttgagt 21000 acgagtcact cctgcgccgt agcgccattg
cttcttcccc cgaccgctgt ataacgctgg 21060 aaaagtccac ccaaagcgta
caggggccca actcggccgc ctgtggacta ttctgctgca 21120 tgtttctcca
cgcctttgcc aactggcccc aaactcccat ggatcacaac cccaccatga 21180
accttattac cggggtaccc aactccatgc tcaacagtcc ccaggtacag cccaccctgc
21240 gtcgcaacca ggaacagctc tacagcttcc tggagcgcca ctcgccctac
ttccgcagcc 21300 acagtgcgca gattaggagc gccacttctt tttgtcactt
gaaaaacatg taaaaataat 21360 gtactagaga cactttcaat aaaggcaaat
gcttttattt gtacactctc gggtgattat 21420 ttacccccac ccttgccgtc
tgcgccgttt aaaaatcaaa ggggttctgc cgcgcatcgc 21480 tatgcgccac
tggcagggac acgttgcgat actggtgttt agtgctccac ttaaactcag 21540
gcacaaccat ccgcggcagc tcggtgaagt tttcactcca caggctgcgc accatcacca
21600 acgcgtttag caggtcgggc gccgatatct tgaagtcgca gttggggcct
ccgccctgcg 21660 cgcgcgagtt gcgatacaca gggttgcagc actggaacac
tatcagcgcc gggtggtgca 21720 cgctggccag cacgctcttg tcggagatca
gatccgcgtc caggtcctcc gcgttgctca 21780 gggcgaacgg agtcaacttt
ggtagctgcc ttcccaaaaa gggcgcgtgc ccaggctttg 21840 agttgcactc
gcaccgtagt ggcatcaaaa ggtgaccgtg cccggtctgg gcgttaggat 21900
acagcgcctg cataaaagcc ttgatctgct taaaagccac ctgagccttt gcgccttcag
21960 agaagaacat gccgcaagac ttgccggaaa actgattggc cggacaggcc
gcgtcgtgca 22020 cgcagcacct tgcgtcggtg ttggagatct gcaccacatt
tcggccccac cggttcttca 22080 cgatcttggc cttgctagac tgctccttca
gcgcgcgctg cccgttttcg ctcgtcacat 22140 ccatttcaat cacgtgctcc
ttatttatca taatgcttcc gtgtagacac ttaagctcgc 22200 cttcgatctc
agcgcagcgg tgcagccaca acgcgcagcc cgtgggctcg tgatgcttgt 22260
aggtcacctc tgcaaacgac tgcaggtacg cctgcaggaa tcgccccatc atcgtcacaa
22320 aggtcttgtt gctggtgaag gtcagctgca acccgcggtg ctcctcgttc
agccaggtct 22380 tgcatacggc cgccagagct tccacttggt caggcagtag
tttgaagttc gcctttagat 22440 cgttatccac gtggtacttg tccatcagcg
cgcgcgcagc ctccatgccc ttctcccacg 22500 cagacacgat cggcacactc
agcgggttca tcaccgtaat ttcactttcc gcttcgctgg 22560 gctcttcctc
ttcctcttgc gtccgcatac cacgcgccac tgggtcgtct tcattcagcc 22620
gccgcactgt gcgcttacct cctttgccat gcttgattag caccggtggg ttgctgaaac
22680 ccaccatttg tagcgccaca tcttctcttt cttcctcgct gtccacgatt
acctctggtg 22740 atggcgggcg ctcgggcttg ggagaagggc gcttcttttt
cttcttgggc gcaatggcca 22800 aatccgccgc cgaggtcgat ggccgcgggc
tgggtgtgcg cggcaccagc gcgtcttgtg 22860 atgagtcttc ctcgtcctcg
gactcgatac gccgcctcat ccgctttttt gggggcgccc 22920 ggggaggcgg
cggcgacggg gacggggacg acacgtcctc catggttggg ggacgtcgcg 22980
ccgcaccgcg tccgcgctcg ggggtggttt cgcgctgctc ctcttcccga ctggccattt
23040 ccttctccta taggcagaaa aagatcatgg agtcagtcga gaagaaggac
agcctaaccg 23100 ccccctctga gttcgccacc accgcctcca ccgatgccgc
caacgcgcct accaccttcc 23160 ccgtcgaggc acccccgctt gaggaggagg
aagtgattat cgagcaggac ccaggttttg 23220 taagcgaaga cgacgaggac
cgctcagtac caacagagga taaaaagcaa gaccaggaca 23280 acgcagaggc
aaacgaggaa caagtcgggc ggggggacga aaggcatggc gactacctag 23340
atgtgggaga cgacgtgctg ttgaagcatc tgcagcgcca gtgcgccatt atctgcgacg
23400 cgttgcaaga gcgcagcgat gtgcccctcg ccatagcgga tgtcagcctt
gcctacgaac 23460 gccacctatt ctcaccgcgc gtacccccca aacgccaaga
aaacggcaca tgcgagccca 23520 acccgcgcct caacttctac cccgtatttg
ccgtgccaga ggtgcttgcc acctatcaca 23580 tctttttcca aaactgcaag
atacccctat cctgccgtgc caaccgcagc cgagcggaca 23640 agcagctggc
cttgcggcag ggcgctgtca tacctgatat cgcctcgctc aacgaagtgc 23700
caaaaatctt tgagggtctt ggacgcgacg agaagcgcgc ggcaaacgct ctgcaacagg
23760 aaaacagcga aaatgaaagt cactctggag tgttggtgga actcgagggt
gacaacgcgc 23820 gcctagccgt actaaaacgc agcatcgagg tcacccactt
tgcctacccg gcacttaacc 23880 taccccccaa ggtcatgagc acagtcatga
gtgagctgat cgtgcgccgt gcgcagcccc 23940 tggagaggga tgcaaatttg
caagaacaaa cagaggaggg cctacccgca gttggcgacg 24000 agcagctagc
gcgctggctt caaacgcgcg agcctgccga cttggaggag cgacgcaaac 24060
taatgatggc cgcagtgctc gttaccgtgg agcttgagtg catgcagcgg ttctttgctg
24120 acccggagat gcagcgcaag ctagaggaaa cattgcacta cacctttcga
cagggctacg 24180 tacgccaggc ctgcaagatc tccaacgtgg agctctgcaa
cctggtctcc taccttggaa 24240 ttttgcacga aaaccgcctt gggcaaaacg
tgcttcattc cacgctcaag ggcgaggcgc 24300 gccgcgacta cgtccgcgac
tgcgtttact tatttctatg ctacacctgg cagacggcca 24360 tgggcgtttg
gcagcagtgc ttggaggagt gcaacctcaa ggagctgcag aaactgctaa 24420
agcaaaactt gaaggaccta tggacggcct tcaacgagcg ctccgtggcc gcgcacctgg
24480 cggacatcat tttccccgaa cgcctgctta aaaccctgca acagggtctg
ccagacttca 24540 ccagtcaaag catgttgcag aactttagga actttatcct
agagcgctca ggaatcttgc 24600 ccgccacctg ctgtgcactt cctagcgact
ttgtgcccat taagtaccgc gaatgccctc 24660 cgccgctttg gggccactgc
taccttctgc agctagccaa ctaccttgcc taccactctg 24720 acataatgga
agacgtgagc ggtgacggtc tactggagtg tcactgtcgc tgcaacctat 24780
gcaccccgca ccgctccctg gtttgcaatt cgcagctgct taacgaaagt caaattatcg
24840 gtacctttga gctgcagggt ccctcgcctg acgaaaagtc cgcggctccg
gggttgaaac 24900 tcactccggg gctgtggacg tcggcttacc ttcgcaaatt
tgtacctgag gactaccacg 24960 cccacgagat taggttctac gaagaccaat
cccgcccgcc aaatgcggag cttaccgcct 25020 gcgtcattac ccagggccac
attcttggcc aattgcaagc catcaacaaa gcccgccaag 25080 agtttctgct
acgaaaggga cggggggttt acttggaccc ccagtccggc gaggagctca 25140
acccaatccc cccgccgccg cagccctatc agcagcagcc gcgggccctt gcttcccagg
25200 atggcaccca aaaagaagct gcagctgccg ccgccaccca cggacgagga
ggaatactgg 25260 gacagtcagg cagaggaggt tttggacgag gaggaggagg
acatgatgga agactgggag 25320 agcctagacg aggaagcttc cgaggtcgaa
gaggtgtcag acgaaacacc gtcaccctcg 25380 gtcgcattcc cctcgccggc
gccccagaaa tcggcaaccg gttccagcat ggctacaacc 25440 tccgctcctc
aggcgccgcc ggcactgccc gttcgccgac ccaaccgtag atgggacacc 25500
actggaacca gggccggtaa gtccaagcag ccgccgccgt tagcccaaga gcaacaacag
25560 cgccaaggct accgctcatg gcgcgggcac aagaacgcca tagttgcttg
cttgcaagac 25620 tgtgggggca acatctcctt cgcccgccgc tttcttctct
accatcacgg cgtggccttc 25680 ccccgtaaca tcctgcatta ctaccgtcat
ctctacagcc catactgcac cggcggcagc 25740 ggcagcggca gcaacagcag
cggccacaca gaagcaaagg cgaccggata gcaagactct 25800 gacaaagccc
aagaaatcca cagcggcggc agcagcagga ggaggagcgc tgcgtctggc 25860
gcccaacgaa cccgtatcga cccgcgagct tagaaacagg atttttccca ctctgtatgc
25920 tatatttcaa cagagcaggg gccaagaaca agagctgaaa ataaaaaaca
ggtctctgcg 25980 atccctcacc cgcagctgcc tgtatcacaa aagcgaagat
cagcttcggc gcacgctgga 26040 agacgcggag gctctcttca gtaaatactg
cgcgctgact cttaaggact agtttcgcgc 26100 cctttctcaa atttaagcgc
gaaaactacg tcatctccag cggccacacc cggcgccagc 26160 acctgtcgtc
agcgccatta tgagcaagga aattcccacg ccctacatgt ggagttacca 26220
gccacaaatg ggacttgcgg ctggagctgc ccaagactac tcaacccgaa taaactacat
26280 gagcgcggga ccccacatga tatcccgggt caacggaatc cgcgcccacc
gaaaccgaat 26340 tctcttggaa caggcggcta ttaccaccac acctcgtaat
aaccttaatc cccgtagttg 26400 gcccgctgcc ctggtgtacc aggaaagtcc
cgctcccacc actgtggtac ttcccagaga 26460 cgcccaggcc gaagttcaga
tgactaactc aggggcgcag cttgcgggcg gctttcgtca 26520 cagggtgcgg
tcgcccgggc agggtataac tcacctgaca atcagagggc gaggtattca 26580
gctcaacgac gagtcggtga gctcctcgct tggtctccgt ccggacggga catttcagat
26640 cggcggcgcc ggccgctctt cattcacgcc tcgtcaggca atcctaactc
tgcagacctc 26700 gtcctctgag ccgcgctctg gaggcattgg aactctgcaa
tttattgagg agtttgtgcc 26760 atcggtctac tttaacccct tctcgggacc
tcccggccac tatccggatc aatttattcc 26820 taactttgac gcggtaaagg
actcggcgga cggctacgac tgaatgttaa gtggagaggc 26880 agagcaactg
cgcctgaaac acctggtcca ctgtcgccgc cacaagtgct ttgcccgcga 26940
ctccggtgag ttttgctact ttgaattgcc cgaggatcat atcgagggcc cggcgcacgg
27000 cgtccggctt accgcccagg gagagcttgc ccgtagcctg attcgggagt
ttacccagcg 27060 ccccctgcta gttgagcggg acaggggacc ctgtgttctc
actgtgattt gcaactgtcc 27120 taaccctgga ttacatcaag atctttgttg
ccatctctgt gctgagtata ataaatacag 27180 aaattaaaat atactggggc
tcctatcgcc atcctgtaaa cgccaccgtc ttcacccgcc 27240 caagcaaacc
aaggcgaacc ttacctggta cttttaacat ctctccctct gtgatttaca 27300
acagtttcaa cccagacgga gtgagtctac gagagaacct ctccgagctc agctactcca
27360 tcagaaaaaa caccaccctc cttacctgcc gggaacgtac gagtgcgtca
ccggccgctg 27420 caccacacct accgcctgac cgtaaaccag actttttccg
gacagacctc aataactctg 27480 tttaccagaa caggaggtga gcttagaaaa
cccttagggt attaggccaa aggcgcagct 27540 actgtggggt ttatgaacaa
ttcaagcaac tctacgggct attctaattc aggtttctct 27600 agaaatggac
ggaattatta cagagcagcg cctgctagaa agacgcaggg cagcggccga 27660
gcaacagcgc atgaatcaag agctccaaga catggttaac ttgcaccagt gcaaaagggg
27720 tatcttttgt ctggtaaagc aggccaaagt cacctacgac agtaatacca
ccggacaccg 27780 ccttagctac aagttgccaa ccaagcgtca gaaattggtg
gtcatggtgg gagaaaagcc 27840 cattaccata actcagcact cggtagaaac
cgaaggctgc attcactcac cttgtcaagg 27900 acctgaggat ctctgcaccc
ttattaagac cctgtgcggt ctcaaagatc ttattccctt 27960 taactaataa
aaaaaaataa taaagcatca cttacttaaa atcagttagc aaatttctgt 28020
ccagtttatt cagcagcacc tccttgccct cctcccagct ctggtattgc agcttcctcc
28080 tggctgcaaa ctttctccac aatctaaatg gaatgtcagt ttcctcctgt
tcctgtccat 28140 ccgcacccac tatcttcatg ttgttgcaga tgaagcgcgc
aagaccgtct gaagatacct 28200 tcaaccccgt gtatccatat gacacggaaa
ccggtcctcc aactgtgcct tttcttactc 28260 ctccctttgt atcccccaat
gggtttcaag agagtccccc tggggtactc tctttgcgcc 28320 tatccgaacc
tctagttacc tccaatggca tgcttgcgct caaaatgggc aacggcctct 28380
ctctggacga ggccggcaac cttacctccc aaaatgtaac cactgtgagc ccacctctca
28440 aaaaaaccaa gtcaaacata aacctggaaa tatctgcacc cctcacagtt
acctcagaag 28500 ccctaactgt ggctgccgcc gcacctctaa tggtcgcggg
caacacactc accatgcaat 28560 cacaggcccc gctaaccgtg cacgactcca
aacttagcat tgccacccaa ggacccctca 28620 cagtgtcaga aggaaagcta
gccctgcaaa catcaggccc cctcaccacc accgatagca 28680 gtacccttac
tatcactgcc tcaccccctc taactactgc cactggtagc ttgggcattg 28740
acttgaaaga gcccatttat acacaaaatg gaaaactagg actaaagtac ggggctcctt
28800 tgcatgtaac agacgaccta aacactttga ccgtagcaac tggtccaggt
gtgactatta 28860 ataatacttc cttgcaaact aaagttactg gagccttggg
ttttgattca caaggcaata 28920 tgcaacttaa tgtagcagga ggactaagga
ttgattctca aaacagacgc cttatacttg 28980 atgttagtta tccgtttgat
gctcaaaacc aactaaatct aagactagga cagggccctc 29040 tttttataaa
ctcagcccac aacttggata ttaactacaa caaaggcctt tacttgttta 29100
cagcttcaaa caattccaaa aagcttgagg ttaacctaag cactgccaag gggttgatgt
29160 ttgacgctac agccatagcc attaatgcag gagatgggct tgaatttggt
tcacctaatg 29220 caccaaacac aaatcccctc aaaacaaaaa ttggccatgg
cctagaattt gattcaaaca 29280 aggctatggt tcctaaacta ggaactggcc
ttagttttga cagcacaggt gccattacag 29340 taggaaacaa aaataatgat
aagctaactt tgtggaccac accagctcca tctcctaact 29400 gtagactaaa
tgcagagaaa gatgctaaac tcactttggt cttaacaaaa tgtggcagtc 29460
aaatacttgc tacagtttca gttttggctg ttaaaggcag tttggctcca atatctggaa
29520 cagttcaaag tgctcatctt attataagat ttgacgaaaa tggagtgcta
ctaaacaatt 29580 ccttcctgga cccagaatat tggaacttta gaaatggaga
tcttactgaa ggcacagcct 29640 atacaaacgc tgttggattt atgcctaacc
tatcagctta tccaaaatct cacggtaaaa 29700 ctgccaaaag taacattgtc
agtcaagttt acttaaacgg agacaaaact aaacctgtaa 29760 cactaaccat
tacactaaac ggtacacagg aaacaggaga cacaactcca agtgcatact 29820
ctatgtcatt ttcatgggac tggtctggcc acaactacat taatgaaata tttgccacat
29880 cctcttacac tttttcatac attgcccaag aataaagaat cgtttgtgtt
atgtttcaac 29940 gtgtttattt ttcaattgcc cgggatcggt gatcaccgat
ccagacatga taagatacat 30000 tgatgagttt ggacaaacca caactagaat
gcagtgaaaa aaatgcttta tttgtgaaat 30060 ttgtgatgct
attgctttat ttgtaaccat tataagctgc aataaacaag ttcccggatc 30120
gcgatccggc ccgaggctgt agccgacgat ggtgcgccag gagagttgtt gattcattgt
30180 ttgcctccct gctgcggttt ttcaccgaag ttcatgccag tccagcgttt
ttgcagcaga 30240 aaagccgccg acttcggttt gcggtcgcga gtgaagatcc
ctttcttgtt accgccaacg 30300 cgcaatatgc cttgcgaggt cgcaaaatcg
gcgaaattcc atacctgttc accgacgacg 30360 gcgctgacgc gatcaaagac
gcggtgatac atatccagcc atgcacactg atactcttca 30420 ctccacatgt
cggtgtacat tgagtgcagc ccggctaacg tatccacgcc gtattcggtg 30480
atgataatcg gctgatgcag tttctcctgc caggccagaa gttctttttc cagtaccttc
30540 tctgccgttt ccaaatcgcc gctttggaca taccatccgt aataacggtt
caggcacagc 30600 acatcaaaga gatcgctgat ggtatcggtg tgagcgtcgc
agaacattac attgacgcag 30660 gtgatcggac gcgtcgggtc gagtttacgc
gttgcttccg ccagtggcgc gaaatattcc 30720 cgtgcacctt gcggacgggt
atccggttcg ttggcaatac tccacatcac cacgcttggg 30780 tggtttttgt
cacgcgctat cagctcttta atcgcctgta agtgcgcttg ctgagtttcc 30840
ccgttgactg cctcttcgct gtacagttct ttcggcttgt tgcccgcttc gaaaccaatg
30900 cctaaagaga ggttaaagcc gacagcagca gtttcatcaa tcaccacgat
gccatgttca 30960 tctgcccagt cgagcatctc ttcagcgtaa gggtaatgcg
aggtacggta ggagttggcc 31020 ccaatccagt ccattaatgc gtggtcgtgc
accatcagca cgttatcgaa tcctttgcca 31080 cgcaagtccg catcttcatg
acgaccaaag ccagtaaagt agaacggttt gtggttaatc 31140 aggaactgtt
cgcccttcac tgccactgac cggatgccga cgcgaagcgg gtagatatca 31200
cactctgtct ggcttttggc tgtgacgcac agttcataga gataaccttc acccggttgc
31260 cagaggtgcg gattcaccac ttgcaaagtc ccgctagtgc cttgtccagt
tgcaaccacc 31320 tgttgatccg catcacgcag ttcaacgctg acatcaccat
tggccaccac ctgccagtca 31380 acagacgcgt ggttacagtc ttgcgcgaca
tgcgtcacca cggtgatatc gtccacccag 31440 gtgttcggcg tggtgtagag
cattacgctg cgatggattc cggcatagtt aaagaaatca 31500 tggaagtaag
actgcttttt cttgccgttt tcgtcggtaa tcaccattcc cggcgggata 31560
gtctgccagt tcagttcgtt gttcacacaa acggtgatac gtacactttt cccggcaata
31620 acatacggcg tgacatcggc ttcaaatggc gtatagccgc cctgatgctc
catcacttcc 31680 tgattattga cccacacttt gccgtaatga gtgaccgcat
cgaaacgcag cacgatacgc 31740 tggcctgccc aacctttcgg tataaagact
tcgcgctgat accagacgtt gcccgcataa 31800 ttacgaatat ctgcatcggc
gaactgatcg ttaaaactgc ctggcacagc aattgcccgg 31860 ctttcttgta
acgcgctttc ccaccaacgc tgatcaattc cacagttttc gcgatccaga 31920
ctgaatgccc acaggccgtc gagttttttg atttcacggg ttggggtttc tacaggacgg
31980 accatgcgtt cgacctttct cttctttttt gggcccatga tggcagatcc
gtatagtgag 32040 tcgtattagc tggttctttc cgcctcagaa gccatagagc
ccaccgcatc cccagcatgc 32100 ctgctattgt cttcccaatc ctcccccttg
ctgtcctgcc ccaccccacc ccccagaata 32160 gaatgacacc tactcagaca
atgcgatgca atttcctcat tttattagga aaggacagtg 32220 ggagtggcac
cttccagggt caaggaaggc acgggggagg ggcaaacaac agatggctgg 32280
caactagaag gcacagtcga ggctgatcag cgagctctag atgcatgctc gagcggccgc
32340 cagtgtgatg gatatctgca gaattccagc acactggcgg ccgttactag
tggatccgag 32400 ctcggtaccc ggccgttata acaccactcg acacggcacc
agctcaatca gtcacagtgt 32460 aaaaaagggc caagtgcaga gcgagtatat
ataggactaa aaaatgacgt aacggttaaa 32520 gtccacaaaa aacacccaga
aaaccgcacg cgaacctacg cccagaaacg aaagccaaaa 32580 aacccacaac
ttcctcaaat cgtcacttcc gttttcccac gttacgtcac ttcccatttt 32640
aagaaaacta caattcccaa cacatacaag ttactccgcc ctaaaaccta cgtcacccgc
32700 cccgttccca cgccccgcgc cacgtcacaa actccacccc ctcattatca
tattggcttc 32760 aatccaaaat aaggtatatt attgatgatg ggtcgtta 32798 2
465 DNA DNA encoding tumor necrosis factor-alpha misc_feature SEQ
ID NO2 represents the nucleotides of positions 1469 through 1933 of
SEQ ID NO1 2 tcatcttctc gaaccccgag tgacaagcct gtagcccatg ttgtagcaaa
ccctcaagct 60 gaggggcagc tccagtggct gaaccgccgg gccaatgccc
tcctggccaa tggcgtggag 120 ctgagagata accagctggt ggtgccatca
gagggcctgt acctcatcta ctcccaggtc 180 ctcttcaagg gccaaggctg
cccctccacc catgtgctcc tcacccacac catcagccgc 240 atcgccgtct
cctaccagac caaggtcaac ctcctctctg ccatcaagag cccctgccag 300
agggagaccc cagagggggc tgaggccaag ccctggtatg agcccatcta tctgggaggg
360 gtcttccagc tggagaaggg tgaccgactc agcgctgaga tcaatcggcc
cgactatctc 420 gactttgccg agtctgggca ggtctacttt gggatcattg ccctg
465
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