U.S. patent application number 10/863056 was filed with the patent office on 2005-06-02 for methods and compositions for modulating telomerase reverse transcriptase (tert) expression.
Invention is credited to Andrews, William H., Foster, Christopher A., Fraser, Stephanie, Mohammadpour, Hamid.
Application Number | 20050118149 10/863056 |
Document ID | / |
Family ID | 27397771 |
Filed Date | 2005-06-02 |
United States Patent
Application |
20050118149 |
Kind Code |
A1 |
Andrews, William H. ; et
al. |
June 2, 2005 |
Methods and compositions for modulating telomerase reverse
transcriptase (TERT) expression
Abstract
Methods and compositions are provided for modulating, and
generally upregulating, the expression of telomerase reverse
transcriptase (TERT) by blocking repression of TERT transcription,
e.g., by inhibiting binding of repressor factor to a Site C
repressor binding site located in the TERT minimal promoter. The
subject methods and compositions find use in a variety of different
applications, including the immortalization of cells, the
production of reagents for use in life science research,
therapeutic applications; therapeutic agent screening applications;
and the like. In further describing the subject invention, the
methods and compositions of the invention are described first in
greater detail, followed by a review of the various applications in
which the subject invention finds use.
Inventors: |
Andrews, William H.; (Reno,
NV) ; Foster, Christopher A.; (Carmichael, CA)
; Fraser, Stephanie; (Sparks, NV) ; Mohammadpour,
Hamid; (Reno, NV) |
Correspondence
Address: |
BOZICEVIC, FIELD & FRANCIS LLP
1900 UNIVERSITY AVENUE
SUITE 200
EAST PALO ALTO
CA
94303
US
|
Family ID: |
27397771 |
Appl. No.: |
10/863056 |
Filed: |
June 7, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10863056 |
Jun 7, 2004 |
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10338294 |
Jan 7, 2003 |
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10338294 |
Jan 7, 2003 |
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09932581 |
Aug 17, 2001 |
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6686159 |
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60227865 |
Aug 24, 2000 |
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60230174 |
Sep 1, 2000 |
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60238345 |
Oct 5, 2000 |
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Current U.S.
Class: |
424/93.21 ;
514/44R |
Current CPC
Class: |
G01N 2500/10 20130101;
C12N 2510/04 20130101; A61P 43/00 20180101; C12N 9/1276 20130101;
C07K 16/00 20130101; A61K 38/00 20130101; G01N 2500/20
20130101 |
Class at
Publication: |
424/093.21 ;
514/044 |
International
Class: |
A61K 048/00 |
Claims
1-27. (canceled)
28. A nucleic acid present in other than its natural environment,
wherein said nucleic acid has a nucleotide sequence that is the
same as or substantially identical to the Site C repressor binding
site and said nucleic acid does not include the full minimal TERT
promoter sequence.
29. The nucleic acid according to claim 28, wherein said nucleic
acid comprises no more than about 90 number % of the full sequence
of the hTERT minimal promoter.
30. The nucleic acid according to claim 29, wherein said nucleic
acid comprises no more than about 50 number % of the full sequence
of the minimal hTERT promoter sequence.
31. The nucleic acid according claim 28, wherein said nucleic acid
has a length ranging from about 5 to about 5000 bases.
32. The nucleic acid according to claim 31, wherein said nucleic
acid has a length ranging from about 10 to about 2500 bases.
33. The nucleic acid according to claim 32, wherein said nucleic
acid has a length ranging from about 10 to about 500 bases.
34. The nucleic acid according to claim 33, wherein said nucleic
acid has a length ranging from about 10 to about 50 bases.
35. The nucleic acid according to claim 28, wherein said nucleic
acid is isolated.
36. The nucleic acid according to claim 28, wherein said nucleic
acid has a sequence that is substantially the same as or identical
to a sequence found in a sequence selected from the group
consisting of SEQ ID NOs:01 to 04.
37. An isolated nucleic acid or mimetic thereof that hybridizes
under stringent conditions to the nucleic acid according to claim
28 or its complementary sequence, wherein said isolated nucleic
acid does not include the full TERT minimal promoter sequence.
38. A construct comprising a nucleic acid according to claim
28.
39. The construct according to claim 38, wherein said construct
comprises a TERT promoter.
40. The construct according to claim 39, wherein said construct is
an expression cassette.
41. A double stranded DNA decoy sequence comprising a Site C
repressor binding site.
42. The decoy according to claim 41, wherein said decoy comprises a
sequence selected from the group consisting of SEQ ID NOs: 01 to
04.
43. The decoy according to claim 42, wherein said decoy ranges in
length from about 10 to about 50 bases.
44. A method comprising administering to cells a decoy according to
claim 41.
45. A mammalian cell comprising a telomerase gene modified by
deletion of any of the nucleotides found in a Site C repressor.
46. The cell according to claim 45, wherein said deletion is any of
nucleotides found in a sequence selected from the group consisting
of SEQ ID NOs: 01 to 04.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of
application Ser. No. 10/338,294 filed on Jan. 7, 2003 and now
published as U.S. Published application No. 2003/0171326; which
application is a continuation application of application Ser. No.
09/932,581 filed Aug. 17, 2001, which application, pursuant to 35
U.S.C. .sctn. 119(e), claims priority to the filing dates of the
U.S. Provisional Patent Application Ser. No. 60/227,865 filed Aug.
24, 2000; 60/230,174 filed Sep. 1, 2000 and 60/238,345 filed Oct.
5, 2000, the disclosures of which are herein incorporated by
reference.
INTRODUCTION
[0002] 1. Field of the Invention
[0003] The field of this invention is the telomerase reverse
transcriptase gene, specifically the regulation of the expression
thereof.
[0004] 2. Background of the Invention
[0005] Telomeres, which define the ends of chromosomes, consist of
short, tandemly repeated DNA sequences loosely conserved in
eukaryotes. Human telomeres consist of many kilobases of (TTAGGG)n
together with various associated proteins. Small amounts of these
terminal sequences or telomeric DNA are lost from the tips of the
chromosomes during S phase because of incomplete DNA replication.
Many human cells progressively lose terminal sequence with cell
division, a loss that correlates with the apparent absence of
telomerase in these cells. The resulting telomeric shortening has
been demonstrated to limit cellular lifespan.
[0006] Telomerase is a ribonucleoprotein that synthesizes telomeric
DNA. Human telomerase is made up of two components: (1) an
essential structural RNA (TER) (where the human component is
referred to in the art as hTER); and (2) a catalytic protein
(telomerase reverse transcriptase or TERT) (where the human
component is referred to in the art as hTERT). Telomerase works by
recognizing the 3' end of DNA, e.g., telomeres, and adding multiple
telomeric repeats to its 3' end with the catalytic protein
component, e.g., hTERT, which has polymerase activity, and hTER
which serves as the template for nucleotide incorporation. Of these
two components of the telomerase enzyme, both the catalytic protein
component and the RNA template component are activity-limiting
components.
[0007] Because of its role in cellular senescence and
immortalization, there is much interest in the development of
protocols and compositions for regulating expression of
telomerase.
[0008] Relevant Literature
[0009] U.S. patents of interest include: U.S. Pat. Nos. 6,093,809;
6,054,575; 6,007,989; 5,958,680; 5,858,777. Also of interest are WO
99/33998 and WO 99/35243. Articles of interest include: Cong et
al., Hum. Mol. Genet. (1999) 8:137-142; Crowe et al., Nucleic Acids
Res. (Jul. 1, 2001) 29:2789-2794; Crowe et al., Biochim Biophys
Acta (Mar. 19, 2001) 1518:1-6; Henderson et al., Head Neck (July
2000) 22:347-354; Kim et al., Oncogene (May 10, 2001) 20:2671-82;
Takakura et al., Cancer Res. (1999) 59:551-7; and Yasui et al., J.
Gastroenterol. (2000) 35 Suppl. 12: 111-115. See also GENBANK
accession nos. AF114847 and 128893.
SUMMARY OF THE INVENTION
[0010] Methods and compositions are provided for modulating, and
generally upregulating, the expression of telomerase reverse
transcriptase (TERT) by blocking repression of TERT transcription,
e.g., by inhibiting binding of repressor factor to a Site C
repressor binding site located in the TERT minimal promoter, where
in certain embodiments the repressor factor acts in concert with
one or more cofactors in binding to the Site C repressor site to
inhibit the TERT transcription site. The subject methods and
compositions find use in a variety of different applications,
including the immortalization of cells, the production of reagents
for use in life science research, therapeutic applications;
therapeutic agent screening applications; and the like.
BRIEF DESCRIPTION OF THE FIGURES
[0011] FIG. 1 provides the sequence of the minimal Tert Promoter
referenced in the experimental section, below.
[0012] FIG. 2 provides an annotated sequence of the pSSI20 plasmid
referenced in the Experimental Section, below.
[0013] FIG. 3 provides graphical results of the fine mapping
analysis experiment reported in the Experimental Section,
below.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
[0014] Methods and compositions are provided for modulating, and
generally upregulating, the expression of telomerase reverse
transcriptase (TERT) by blocking repression of TERT transcription,
e.g., by inhibiting binding of repressor factor to a Site C
repressor binding site located in the TERT minimal promoter, where
in certain embodiments the repressor factor acts in concert with
one or more cofactors in binding to the Site C repressor site to
inhibit the TERT transcription site. The subject methods and
compositions find use in a variety of different applications,
including the immortalization of cells, the production of reagents
for use in life science research, therapeutic applications;
therapeutic agent screening applications; and the like. In further
describing the subject invention, the methods and compositions of
the invention are described first in greater detail, followed by a
review of the various applications in which the subject invention
finds use.
[0015] Before the subject invention is described further, it is to
be understood that the invention is not limited to the particular
embodiments of the invention described below, as variations of the
particular embodiments may be made and still fall within the scope
of the appended claims. It is also to be understood that the
terminology employed is for the purpose of describing particular
embodiments, and is not intended to be limiting. Instead, the scope
of the present invention will be established by the appended
claims.
[0016] In this specification and the appended claims, the singular
forms "a," "an" and "the" include plural reference unless the
context clearly dictates otherwise. Unless defined otherwise, all
technical and scientific terms used herein have the same meaning as
commonly understood to one of ordinary skill in the art to which
this invention belongs.
[0017] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range, and any other stated or intervening
value in that stated range, is encompassed within the invention.
The upper and lower limits of these smaller ranges may
independently be included in the smaller ranges, and are also
encompassed within the invention, subject to any specifically
excluded limit in the stated range. Where the stated range includes
one or both of the limits, ranges excluding either or both of those
included limits are also included in the invention.
[0018] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which this invention belongs. Although
any methods, devices and materials similar or equivalent to those
described herein can be used in the practice or testing of the
invention, the preferred methods, devices and materials are now
described.
[0019] Methods
[0020] As summarized above, the subject invention provides methods
and compositions for modulating expression of TERT. In the subject
methods, TERT expression repression is modulated by modulating the
TERT expression repression activity of a Site C repressor binding
site located in the TERT minimal promoter, where modulating
includes both increasing and decreasing the expression repressive
activity of the Site C repressor binding site. As such, in certain
embodiments, methods of increasing expression of TERT are provided,
while in other embodiments, methods of decreasing expression of
TERT are provided, where in both embodiments the modulation of
expression is accomplished by modulating the repressor activity of
the Site C repressor site.
[0021] Site C Repressor Site
[0022] The Site C repressor site whose activity is modulated in the
subject methods comprises a sequence of nucleotide residues that is
bound by an E2F protein, or at least an E2F DNA binding domain of
an E2F protein. E2F proteins to which the subject Site C repressor
site binds include, but are not limited to: E2F-1, E2F-2, E2F-3,
E2F-4, E2F-5 and E2F-6.
[0023] The target Site C repressor site typically ranges in length
from about 1 base, usually at least about 5 bases and more usually
at least about 15 bases, to a length of about 25 bases or longer,
e.g., 50, 75 or 100, etc. In many embodiments, the length of the
target Site C repressor site/domain ranges in length from about 1
to about 50 bases, usually from about 5 to about 45 bases.
[0024] In many embodiments, the target Site C site has a sequence
found in a limited region of the human tert minimal promoter, where
this limited region typically ranges from about -40 to about -90,
usually from about -45 to about -85 and more usually from about -45
to about -80 relative to the "A" of the telomerase ATG codon.
[0025] Of particular interest in certain embodiments is a nucleic
acid having a sequence found in SEQ ID NO:01 (e.g., a sequence
range of at least about 2, usually at least about 5 and often at
least about 10, 20, 25, 30 or more bases up to about 45 to 50
bases, where, in certain embodiments, the target Site C domain will
have a sequence that is identical to a sequence of SEQ ID NO:01.
SEQ ID NO:01 has the following sequence:
1 (SEQ ID NO:01) GGCCCCGCCCTCTCCTCGCGGCGCGAGTTTCAGGCAGCGCT
[0026] In certain embodiments, the target Site C site includes the
sequence of -69 to -57 of the human TERT minimal promoter. In other
words, the sequence of the Site C site is:
2 GGCGCGAGTTTCA. (SEQ ID NO:02)
[0027] In certain embodiments, the target Site C site includes the
sequence of -67 to -58 of the human TERT minimal promoter. In other
words, the sequence of the Site C site is:
3 CGCGAGTTTC. (SEQ ID NO:03)
[0028] In certain embodiments, the target Site C site includes the
sequence of -69 to -49 of the human TERT minimal promoter. In other
words, the sequence of the Site C site is:
4 GGCGCGAGTTTCAGGCAGCGC. (SEQ ID NO:04)
[0029] Also of interest are Site C sites that have a sequence that
is substantially the same as, or identical to, the Site C repressor
binding site sequences as described above, e.g., SEQ ID NOs: 01 to
04. A given sequence is considered to be substantially similar to
this particular sequence if it shares high sequence similarity with
the above described specific sequences, e.g. at least 75% sequence
identity, usually at least 90%, more usually at least 95% sequence
identity with the above specific sequences. Sequence similarity is
calculated based on a reference sequence, which may be a subset of
a larger sequence. A reference sequence will usually be at least
about 18 nt long, more usually at least about 30 nt long, and may
extend to the complete sequence that is being compared. Algorithms
for sequence analysis are known in the art, such as BLAST,
described in Altschul et al. (1990), J. Mol. Biol. 215:403-10
(using default settings, i.e. parameters w=4 and T=17). Of
particular interest in certain embodiments are nucleic acids of
substantially the same length as the specific nucleic acid
identified above, where by substantially the same length is meant
that any difference in length does not exceed about 20 number %,
usually does not exceed about 10 number % and more usually does not
exceed about 5 number %; and have sequence identity to this
sequence of at least about 90%, usually at least about 95% and more
usually at least about 99% over the entire length of the nucleic
acid. Also of interest are nucleic acids that represent a modified
or altered Site C site, e.g., where the site includes one or more
deletions or substitutions as compared to the above specific Site C
sequences, including a deletion or substitution of a portion of the
Site C repressor binding site, e.g., a deletion or substitution of
at least one nucleotide.
[0030] Modulating TERT Expression
[0031] The subject invention provides methods of modulating,
including both enhancing and repressing, TERT expression. As such,
methods of both increasing and decreasing TERT expression are
provided. In many embodiments, such methods are methods of
modulating the binding interaction and resultant Site C TERT
expression repression activity between a Site C site in a minimal
TERT promoter and a Site C repressor protein, where in many
embodiments the Site C repressor protein is a protein having an E2F
DNA binding domain, particularly a Site C E2F DNA binding domain.
As such, included are methods of either enhancing or inhibiting
binding of Site C repressor protein to a TERT minimal promoter Site
C site.
[0032] As indicated above, the Site C repressor protein whose
interaction with the Site C repressor site is modulated in the
subject methods is a protein that binds to the Site C repressor
site and, in so binding, inhibits TERT expression. In many
embodiments, the Site C repressor protein is a protein that binds
to the Site C site via an E2F DNA binding domain present on the
repressor protein, i.e., that is part of the repressor protein. In
certain embodiments, the target Site C repressor proteins are
proteins that include a DNA binding domain having a sequence of
residues according to the following formula, where X is any
residue:
R--(X).sub.38--R--R--X--Y
[0033] In certain embodiments, the target Site C repressor proteins
are proteins that include a DNA binding domain that has an amino
acid sequence that is at least homologous to the amino acid
sequence of the DNA binding domain of either E2F-1 or E2F-4. The
amino acid sequence of the DNA binding domain of E2F-1 is:
5 GRGRHPGKGVKSPGEKSRYETSLNLTTKRFLELLSHSADGVVDLNWAAEVLKV (SEQ ID
NO:05) QKRRIYDITNVLEGIQLIA KKSKNHIQWLGSH.
[0034] The amino acid sequence of the DNA binding domain of E2F-4
is:
6 PPGTPSRHEKSLGLLTTKFVSLLQEAKDGVLDLKLAADTLAVRQKRRIYDITNVLE (SEQ ID
NO:06) GIGLIEKKSKNSIQWK GVGP.
[0035] By at least homologous is meant that the target Site C
repressor protein has a DNA binding domain which includes an amino
acid sequence that has at least 20%, usually at least 25% sequence
identity with at least one of the specific E2F binding domains
provided above, where sequence identity for this particular purpose
is measured using the BLAST compare two sequences program available
on the NCBI website using default settings.
[0036] As such, in certain embodiments, target repressor proteins
are E2F proteins. Target E2F proteins of interest include, but are
not limited to: E2F-1, E2F-2, E2F-3, E2F-4, E2F-5 and E2F-6; where
in certain embodiments, E2F-6 is the target protein of interest. In
yet other embodiments, the target Site C repressor protein is not
an E2F protein, but is instead a protein that includes an E2F DNA
binding site, as described above, or homologue thereof. In certain
embodiments, the target Site C repressor protein acts in concert
with one or more cofactors in binding to the Site C repressor site
to inhibit the TERT transcription site. For example, in certain
embodiments the Site C repressor protein's repressive activity upon
binding to the Site C sequence is modulated by its interaction with
one or more additional cofactors, in a manner analogous to the
manner in which E2F's 1-5 are known to be converted from activators
to repressors by binding to a cofactor from the Retinoblastoma (RB)
family of proteins, including pRB, p107, or p130, as reviewed in:
"The Regulation of E2F by pRB-Family proteins", N. Dyson; Genes
Dev, 12, p 2245-62 (1998).
[0037] In modulating TERT expression, the interaction between the
Site C repressor site and its repressor protein can be modified
directly or indirectly. An example of direct modification of this
interaction is where the binding of the repress or protein to the
target sequence is modified by an agent that directly changes how
the repressor protein binds to the Site C sequence, e.g., by
occupying the DNA binding site of the repressor protein, by binding
to the Site C sequence thereby preventing its binding to the
repressor protein, etc. An example of indirect modification is
modification/modulation of the Site C repressive activity via
disruption of a binding interaction between the repressor protein
and one or more cofactors (or further upstream in the chain of
interactions, such as cofactors that interact with the Site C
binding protein to make it either a repressor or activator, as
described above) such that the repressive activity is modulated, by
modification/alteration of the Site C DNA binding sequence such
that binding to the repressor protein is modulated, etc.
[0038] Enhancing TERT Expression
[0039] Methods are provided for enhancing TERT expression. By
enhancing TERT expression is meant that the expression level of the
TERT coding sequence is increased by at least about 2 fold, usually
by at least about 5 fold and sometimes by at least 25, 50, 100 fold
and in particular about 300 fold or higher, as compared to a
control, i.e., expression from an expression system that is not
subjected to the methods of the present invention. Alternatively,
in cases where expression of the TERT gene is so low that it is
undetectable, expression of the TERT gene is considered to be
enhanced if expression is increased to a level that is easily
detectable.
[0040] In these methods, Site C repression of TERT expression is
inhibited. By inhibited is meant that the repressive activity of
the TERT Site C repressor binding site/repressor protein
interaction with respect to TERT expression is decreased by a
factor sufficient to at least provide for the desired enhanced
level of TERT expression, as described above. Inhibition of Site C
transcription repression may be accomplished in a number of ways,
where representative protocols for inhibiting this TERT expression
repression are now provided.
[0041] One representative method of inhibiting repression of
transcription is to employ double-stranded, i.e., duplex,
oligonucleotide decoys for the Site C repressor protein, which bind
to the Site C repressor protein and thereby prevent the Site C
repressor protein binding to its target Site C site in the TERT
promoter, e.g., the Site C site of the TERT minimal promoter. These
duplex oligonucleotide decoys have at least that portion of the
sequence of the TERT Site C site required to bind to the Site C
repressor protein and thereby prevent its binding to the Site C
site. In many embodiments, the subject decoy nucleic acid molecules
include a sequence of nucleotides that is the same as a sequence
found in SEQ ID NOs: 01 to 04. In other embodiments, the subject
decoy nucleic acid molecules include a sequence of nucleotides that
is substantially the same as or identical to a sequence found in
SEQ ID NOs: 01 to 04; where the terms substantially the same as and
identical thereto in relation to nucleic acids are defined below.
In many embodiments, the length of these duplex oligonucleotide
decoys ranges from about 5 to about 5000, usually from about 5 to
about 500 and more usually from about 10 to about 50 bases. In
using such oligonucleotide decoys, the decoys are placed into the
environment of the Site C site and its Site C repressor protein,
resulting in de-repression of the transcription and expression of
the TERT coding sequence. Oligonucleotide decoys and methods for
their use and administration are further described in general terms
in Morishita et al., Circ Res (1998) 82 (10):1023-8. These
oligonucleotide decoys generally include a TERT Site C repressor
binding site recognized by the target Site C repressor protein,
including the specific regions detailed above, where these
particular embodiments include nucleic acid compositions of the
subject invention, as described in greater detail below.
[0042] Instead of the above described decoys, other agents that
disrupt binding of the Site C repressor protein to the target TERT
Site C repressor binding site and thereby inhibit its expression
repression activity may be employed. Other agents of interest
include, among other types of agents, small molecules that bind to
the Site C repressor protein and inhibit its binding to the Site C
repressor region. Alternatively, agents that bind to the Site C
sequence and inhibit its binding to the Site C repressor protein
are of interest. Alternatively, agents that disrupt Site C
repressor protein-protein interactions with cofactors, e.g.,
cofactor binding, and thereby inhibit Site C repression are of
interest.
[0043] Naturally occurring or synthetic small molecule compounds of
interest include numerous chemical classes, though typically they
are organic molecules, preferably small organic compounds having a
molecular weight of more than 50 and less than about 2,500 daltons.
Candidate agents comprise functional groups necessary for
structural interaction with proteins, particularly hydrogen
bonding, and typically include at least an amine, carbonyl,
hydroxyl or carboxyl group, preferably at least two of the
functional chemical groups. The candidate agents often comprise
cyclical carbon or heterocyclic structures and/or aromatic or
polyaromatic structures substituted with one or more of the above
functional groups. Candidate agents are also found among
biomolecules including peptides, saccharides, fatty acids,
steroids, purines, pyrimidines, derivatives, structural analogs or
combinations thereof. Such molecules may be identified, among other
ways, by employing the screening protocols described below. Small
molecule agents of particular interest include pyrrole-imidazole
polyamides, analogous to those described in Dickinson et al.,
Biochemistry 1999 Aug 17;38(33):10801-7. Other agents include
"designer" DNA binding proteins that bind Site C (without causing
repression) and prevent the Site C repressor protein from
binding.
[0044] In yet other embodiments, expression of the Site C repressor
protein is inhibited. Inhibition of Site C repressor protein
expression may be accomplished using any convenient means,
including administration of an agent that inhibits Site C repressor
expression (e.g., antisense agents), inactivation of the Site C
repressor gene, e.g., through recombinant techniques, etc.
[0045] For example, where the Site C repressor protein is an E2F
protein, e.g., E2F-6 or a homologue thereof, antisense molecules
can be used to down-regulate expression of the target repressor
protein in cells. The anti-sense reagent may be antisense
oligodeoxynucleotides (ODN), particularly synthetic ODN having
chemical modifications from native nucleic acids, or nucleic acid
constructs that express such anti-sense molecules as RNA. The
antisense sequence is complementary to the mRNA of the targeted
repressor protein, and inhibits expression of the targeted
repressor protein. Antisense molecules inhibit gene expression
through various mechanisms, e.g. by reducing the amount of mRNA
available for translation, through activation of RNAse H, or steric
hindrance. One or a combination of antisense molecules may be
administered, where a combination may comprise multiple different
sequences.
[0046] Antisense molecules may be produced by expression of all or
a part of the target gene sequence in an appropriate vector, where
the transcriptional initiation is oriented such that an antisense
strand is produced as an RNA molecule. Alternatively, the antisense
molecule is a synthetic oligonucleotide. Antisense oligonucleotides
will generally be at least about 7, usually at least about 12, more
usually at least about 20 nucleotides in length, and not more than
about 500, usually not more than about 50, more usually not more
than about 35 nucleotides in length, where the length is governed
by efficiency of inhibition, specificity, including absence of
cross-reactivity, and the like. It has been found that short
oligonucleotides, of from 7 to 8 bases in length, can be strong and
selective inhibitors of gene expression (see Wagner et al. (1996),
Nature Biotechnol. 14:840-844).
[0047] A specific region or regions of the endogenous sense strand
mRNA sequence is chosen to be complemented by the antisense
sequence. Selection of a specific sequence for the oligonucleotide
may use an empirical method, where several candidate sequences are
assayed for inhibition of expression of the target gene in an in
vitro or animal model. A combination of sequences may also be used,
where several regions of the mRNA sequence are selected for
antisense complementation.
[0048] Antisense oligonucleotides may be chemically synthesized by
methods known in the art (see Wagner et al. (1993), supra, and
Milligan et al., supra.) Preferred oligonucleotides are chemically
modified from the native phosphodiester structure, in order to
increase their intracellular stability and binding affinity. A
number of such modifications have been described in the literature,
which alter the chemistry of the backbone, sugars or heterocyclic
bases.
[0049] Among useful changes in the backbone chemistry are
phosphorothioates; phosphorodithioates, where both of the
non-bridging oxygens are substituted with sulfur;
phosphoroamidites; alkyl phosphotriesters and boranophosphates.
Achiral phosphate derivatives include 3'-O'-5'-S-phosphorothioate,
3'-S-5'-O-phosphorothioate, 3'-CH.sub.2-5'-O-phosphonate and
3'-NH-5'-O-phosphoroamidate. Peptide nucleic acids replace the
entire ribose phosphodiester backbone with a peptide linkage. Sugar
modifications are also used to enhance stability and affinity. The
.alpha.-anomer of deoxyribose may be used, where the base is
inverted with respect to the natural .beta.-anomer. The 2'-OH of
the ribose sugar may be altered to form 2'-O-methyl or 2'-O-allyl
sugars, which provides resistance to degradation without comprising
affinity. Modification of the heterocyclic bases must maintain
proper base pairing. Some useful substitutions include deoxyuridine
for deoxythymidine; 5-methyl-2'-deoxycytidine and
5-bromo-2'-deoxycytidine for deoxycytidine.
5-propynyl-2'-deoxyuridine and 5-propynyl-2'-deoxycytidine have
been shown to increase affinity and biological activity when
substituted for deoxythymidine and deoxycytidine, respectively.
[0050] As an alternative to anti-sense inhibitors, catalytic
nucleic acid compounds, e.g. ribozymes, anti-sense conjugates, etc.
may be used to inhibit gene expression. Ribozymes may be
synthesized in vitro and administered to the patient, or may be
encoded on an expression vector, from which the ribozyme is
synthesized in the targeted cell (for example, see International
patent application WO 9523225, and Beigelman et al. (1995), Nucl.
Acids Res. 23:4434-42). Examples of oligonucleotides with catalytic
activity are described in WO 9506764. Conjugates of anti-sense ODN
with a metal complex, e.g. terpyridylCu(II), capable of mediating
mRNA hydrolysis are described in Bashkin et al. (1995), Appl.
Biochem. Biotechnol. 54:43-56.
[0051] In another embodiment, the Site C repressor protein gene is
inactivated so that it no longer expresses a functional repressor
protein. By inactivated is meant that the Site C repressor gene,
e.g., coding sequence and/or regulatory elements thereof, is
genetically modified so that it no longer expresses functional
repressor protein. The alteration or mutation may take a number of
different forms, e.g., through deletion of one or more nucleotide
residues in the repressor region, through exchange of one or more
nucleotide residues in the repressor region, and the like. One
means of making such alterations in the coding sequence is by
homologous recombination. Methods for generating targeted gene
modifications through homologous recombination are known in the
art, including those described in: U.S. Pat. Nos. 6,074,853;
5,998,209; 5,998,144; 5,948,653; 5,925,544; 5,830,698; 5,780,296;
5,776,744; 5,721,367; 5,614,396; 5,612,205; the disclosures of
which are herein incorporated by reference.
[0052] The above described methods of enhancing TERT expression
find use in a number of different applications. In many
applications, the subject methods and compositions are employed to
enhance TERT expression in a cell that endogenously comprises a
TERT gene, e.g. for enhancing expression of hTERT in a normal human
cell in which TERT expression is repressed. The target cell of
these applications is, in many instances, a normal cell, e.g. a
somatic cell. Expression of the TERT gene is considered to be
enhanced if, consistent with the above description, expression is
increased by at least about 2 fold, usually at least about 5 fold
and often 25, 50, 100 fold, 300 fold or higher, as compared to a
control, e.g., an otherwise identical cell not subjected to the
subject methods, or becomes detectable from an initially
undetectable state, as described above.
[0053] A more specific application in which the subject methods
find use is to increase the proliferative capacity of a cell. The
term "proliferative capacity" as used herein refers to the number
of divisions that a cell can undergo, and preferably to the ability
of the target cell to continue to divide where the daughter cells
of such divisions are not transformed, i.e., they maintain normal
response to growth and cell cycle regulation. The subject methods
typically result in an increase in proliferative capacity of at
least about 1.2-2 fold, usually at least about 5 fold and often at
least about 10, 20, 50 fold or even higher, compared to a control.
As such, yet another more specific application in which the subject
methods find use is in the delay of the occurrence of cellular
senescence. By practicing the subject methods, the onset of
cellular senescence may be delayed by a factor of at least about
1.2-2 fold, usually at least about 5 fold and often at least about
10, 20, 50 fold or even higher, compared to a control.
[0054] Methods of Inhibiting TERT Expression
[0055] As mentioned above, also provided are methods for inhibiting
TERT expression, e.g., by enhancing Site C repression of TERT
expression and thereby inhibiting TERT expression. In such methods,
the amount and/or activity of the Site C repressor protein is
increased so as to enhance Site C repressor mediated repression of
TERT expression. A variety of different protocols may be employed
to achieve this result, including administration of an effective
amount of the Site C repressor protein or analog/mimetic thereof,
an agent that enhances expression of Site C repressor protein or an
agent that enhances the activity of the Site C repressor
protein.
[0056] As such, the nucleic acid compositions that encode the Site
C repressor protein find use in situations where one wishes to
enhance the activity of the repressor protein in a host. The
repressor protein genes, gene fragments, or the encoded proteins or
protein fragments are useful in gene therapy to treat disorders in
which inhibition of TERT expression is desired, including those
applications described in greater detail below. Expression vectors
may be used to introduce the gene into a cell. Such vectors
generally have convenient restriction sites located near the
promoter sequence to provide for the insertion of nucleic acid
sequences. Transcription cassettes may be prepared comprising a
transcription initiation region, the target gene or fragment
thereof, and a transcriptional termination region. The
transcription cassettes may be introduced into a variety of
vectors, e.g. plasmid; retrovirus, e.g. lentivirus; adenovirus; and
the like, where the vectors are able to transiently or stably be
maintained in the cells, usually for a period of at least about one
day, more usually for a period of at least about several days to
several weeks.
[0057] The gene or protein may be introduced into tissues or host
cells by any number of routes, including viral infection,
microinjection, or fusion of vesicles. Jet injection may also be
used for intramuscular administration, as described by Furth et al.
(1992), Anal Biochem 205:365-368. The DNA may be coated onto gold
microparticles, and delivered intradermally by a particle
bombardment device, or "gene gun" as described in the literature
(see, for example, Tang et al. (1992), Nature 356:152-154), where
gold microprojectiles are coated with the DNA, then bombarded into
skin cells.
[0058] Therapeutic Applications of TERT Expression Modulation
[0059] The methods find use in a variety of therapeutic
applications in which it is desired to modulate, e.g., increase or
decrease, TERT expression in a target cell or collection of cells,
where the collection of cells may be a whole animal or portion
thereof, e.g., tissue, organ, etc. As such, the target cell(s) may
be a host animal or portion thereof, or may be a therapeutic cell
(or cells) which is to be introduced into a multicellular organism,
e.g., a cell employed in gene therapy. In such methods, an
effective amount of an active agent that modulates TERT expression,
e.g., enhances or decreases TERT expression as desired, is
administered to the target cell or cells, e.g., by contacting the
cells with the agent, by administering the agent to the animal,
etc. By effective amount is meant a dosage sufficient to modulate
TERT expression in the target cell(s), as desired.
[0060] In the subject methods, the active agent(s) may be
administered to the targeted cells using any convenient means
capable of resulting in the desired enhancement of TERT expression.
Thus, the agent can be incorporated into a variety of formulations
for therapeutic administration. More particularly, the agents of
the present invention can be formulated into pharmaceutical
compositions by combination with appropriate, pharmaceutically
acceptable carriers or diluents, and may be formulated into
preparations in solid, semi-solid, liquid or gaseous forms, such as
tablets, capsules, powders, granules, ointments (e.g., skin
creams), solutions, suppositories, injections, inhalants and
aerosols. As such, administration of the agents can be achieved in
various ways, including oral, buccal, rectal, parenteral,
intraperitoneal, intradermal, transdermal, intracheal, etc.,
administration.
[0061] In pharmaceutical dosage forms, the agents may be
administered in the form of their pharmaceutically acceptable
salts, or they may also be used alone or in appropriate
association, as well as in combination, with other pharmaceutically
active compounds. The following methods and excipients are merely
exemplary and are in no way limiting.
[0062] For oral preparations, the agents can be used alone or in
combination with appropriate additives to make tablets, powders,
granules or capsules, for example, with conventional additives,
such as lactose, mannitol, corn starch or potato starch; with
binders, such as crystalline cellulose, cellulose derivatives,
acacia, corn starch or gelatins; with disintegrators, such as corn
starch, potato starch or sodium carboxymethylcellulose; with
lubricants, such as talc or magnesium stearate; and if desired,
with diluents, buffering agents, moistening agents, preservatives
and flavoring agents.
[0063] The agents can be formulated into preparations for injection
by dissolving, suspending or emulsifying them in an aqueous or
nonaqueous solvent, such as vegetable or other similar oils,
synthetic aliphatic acid glycerides, esters of higher aliphatic
acids or propylene glycol; and if desired, with conventional
additives such as solubilizers, isotonic agents, suspending agents,
emulsifying agents, stabilizers and preservatives.
[0064] The agents can be utilized in aerosol formulation to be
administered via inhalation. The compounds of the present invention
can be formulated into pressurized acceptable propellants such as
dichlorodifluoromethane, propane, nitrogen and the like.
[0065] Furthermore, the agents can be made into suppositories by
mixing with a variety of bases such as emulsifying bases or
water-soluble bases. The compounds of the present invention can be
administered rectally via a suppository. The suppository can
include vehicles such as cocoa butter, carbowaxes and polyethylene
glycols, which melt at body temperature, yet are solidified at room
temperature.
[0066] Unit dosage forms for oral or rectal administration such as
syrups, elixirs, and suspensions may be provided wherein each
dosage unit, for example, teaspoonful, tablespoonful, tablet or
suppository, contains a predetermined amount of the composition
containing one or more inhibitors. Similarly, unit dosage forms for
injection or intravenous administration may comprise the
inhibitor(s) in a composition as a solution in sterile water,
normal saline or another pharmaceutically acceptable carrier.
[0067] The term "unit dosage form," as used herein, refers to
physically discrete units suitable as unitary dosages for human and
animal subjects, each unit containing a predetermined quantity of
compounds of the present invention calculated in an amount
sufficient to produce the desired effect in association with a
pharmaceutically acceptable diluent, carrier or vehicle. The
specifications for the novel unit dosage forms of the present
invention depend on the particular compound employed and the effect
to be achieved, and the pharmacodynamics associated with each
compound in the host.
[0068] The pharmaceutically acceptable excipients, such as
vehicles, adjuvants, carriers or diluents, are readily available to
the public. Moreover, pharmaceutically acceptable auxiliary
substances, such as pH adjusting and buffering agents, tonicity
adjusting agents, stabilizers, wetting agents and the like, are
readily available to the public.
[0069] Where the agent is a polypeptide, polynucleotide, analog or
mimetic thereof, e.g. oligonucleotide decoy, it may be introduced
into tissues or host cells by any number of routes, including viral
infection, microinjection, or fusion of vesicles. Jet injection may
also be used for intramuscular administration, as described by
Furth et al. (1992), Anal Biochem 205:365-368. The DNA may be
coated onto gold microparticles, and delivered intradermally by a
particle bombardment device, or "gene gun" as described in the
literature (see, for example, Tang et al. (1992), Nature
356:152-154), where gold microprojectiles are coated with the DNA,
then bombarded into skin cells. For nucleic acid therapeutic
agents, a number of different delivery vehicles find use, including
viral and non-viral vector systems, as are known in the art.
[0070] Those of skill in the art will readily appreciate that dose
levels can vary as a function of the specific compound, the nature
of the delivery vehicle, and the like. Preferred dosages for a
given compound are readily determinable by those of skill in the
art by a variety of means.
[0071] The subject methods find use in the treatment of a variety
of different conditions in which the enhancement of TERT expression
in the host is desired. By treatment is meant that at least an
amelioration of the symptoms associated with the condition
afflicting the host is achieved, where amelioration is used in a
broad sense to refer to at least a reduction in the magnitude of a
parameter, e.g. symptom (such as inflammation), associated with the
condition being treated. As such, treatment also includes
situations where the pathological condition, or at least symptoms
associated therewith, are completely inhibited, e.g. prevented from
happening, or stopped, e.g. terminated, such that the host no
longer suffers from the condition, or at least the symptoms that
characterize the condition.
[0072] A variety of hosts are treatable according to the subject
methods. Generally such hosts are "mammals" or "mammalian," where
these terms are used broadly to describe organisms which are within
the class mammalia, including the orders carnivore (e.g., dogs and
cats), rodentia (e.g., mice, guinea pigs, and rats), and primates
(e.g., humans, chimpanzees, and monkeys). In many embodiments, the
hosts will be humans.
[0073] As indicated above, the subject invention provides methods
of treating disease conditions resulting from a lack of TERT
expression and methods of treating disease conditions resulting
from unwanted TERT expression. Representative disease conditions
for each category are now described in greater detail
separately.
[0074] Treatment of Disease Conditions by Increasing TERT
Expression
[0075] One representative disease condition that may be treated
according to the subject invention is Progeria, or
Hutchinson-Gilford syndrome. This condition is a disease of
shortened telomeres for which no known cure exists. It afflicts
children, who seldom live past their early twenties. In many ways
progeria parallels aging itself. However, these children are born
with short telomeres. Their telomeres don't shorten at a faster
rate; they are just short to begin with. The subject methods can be
used in such conditions to further delay natural telomeric
shortening and/or increase telomeric length, thereby treating this
condition.
[0076] Another specific disease condition in which the subject
methods find use is in immune senescence. The effectiveness of the
immune system decreases with age. Part of this decline is due to
fewer T-lymphocytes in the system, a result of lost replicative
capacity. Many of the remaining T-lymphocytes experience loss of
function as their telomeres shorten and they approach senescence.
The subject methods can be employed to inhibit immune senescence
due to telomere loss. Because hosts with aging immune systems are
at greater risk of developing pneumonia, cellulitis, influenza, and
many other infections, the subject methods reduce morbidity and
mortality due to infections.
[0077] The subject methods also find use in AIDS therapy. HIV, the
virus that causes AIDS, invades white blood cells, particularly CD4
lymphocyte cells, and causes them to reproduce high numbers of the
HIV virus, ultimately killing cells. In response to the loss of
immune cells (typically about a billion per day), the body produces
more CD8 cells to be able to suppress infection. This rapid cell
division accelerates telomere shortening, ultimately hastening
immune senescence of the CD8 cells. Anti-retroviral therapies have
successfully restored the immune systems of AIDS patients, but
survival depends upon the remaining fraction of the patient's aged
T-cells. Once shortened, telomere length has not been naturally
restored within cells. The subject methods can be employed to
restore this length and/or prevent further shortening. As such the
subject methods can spare telomeres and is useful in conjunction
with the anti-retroviral treatments currently available for
HIV.
[0078] Yet another type of disease condition in which the subject
methods find use is cardiovascular disease. The subject methods can
be employed to extend telomere length and replicative capacity of
endothelial cells lining blood vessel walls (DeBono, Heart
80:110-1, 1998). Endothelial cells form the inner lining of blood
vessels and divide and replace themselves in response to stress.
Stresses include high blood pressure, excess cholesterol,
inflammation, and flow stresses at forks in vessels. As endothelial
cells age and can no longer divide sufficiently to replace lost
cells, areas under the endothelial layer become exposed. Exposure
of the underlying vessel wall increases inflammation, the growth of
smooth muscle cells, and the deposition of cholesterol. As a
result, the vessel narrows and becomes scarred and irregular, which
contributes to even more stress on the vessel (Cooper, Cooke and
Dzau, J Gerontol Biol Sci 49: 191-6, 1994). Aging endothelial cells
also produce altered amounts of trophic factors (hormones that
affect the activity of neighboring cells). These too contribute to
increased clotting, proliferation of smooth muscle cells, invasion
by white blood cells, accumulation of cholesterol, and other
changes, many of which lead to plaque formation and clinical
cardiovascular disease (Ibid.). By extending endothelial cell
telomeres, the subject methods can be employed to combat the
stresses contributing to vessel disease. Many heart attacks may be
prevented if endothelial cells were enabled to continue to divide
normally and better maintain cardiac vessels. The occurrence of
strokes caused by the aging of brain blood vessels may also be
significantly reduced by employing the subject methods to help
endothelial cells in the brain blood vessels to continue to divide
and perform their intended function.
[0079] The subject methods also find use in skin rejuvenation. The
skin is the first line of defense of the immune system and shows
the most visible signs of aging (West, Arch Dermatol 130(1):87-95,
1994). As skin ages, it thins, develops wrinkles, discolors, and
heals poorly. Skin cells divide quickly in response to stress and
trauma; but, over time, there are fewer and fewer actively dividing
skin cells. Compounding the loss of replicative capacity in aging
skin is a corresponding loss of support tissues. The number of
blood vessels in the skin decreases with age, reducing the
nutrients that reach the skin. Also, aged immune cells less
effectively fight infection. Nerve cells have fewer branches,
slowing the response to pain and increasing the chance of trauma.
In aged skin, there are also fewer fat cells, increasing
susceptibility to cold and temperature changes. Old skin cells
respond more slowly and less accurately to external signals. They
produce less vitamin D, collagen, and elastin, allowing the
extracellular matrix to deteriorate. As skin thins and loses
pigment with age, more ultraviolet light penetrates and damages
skin. To repair the increasing ultraviolet damage, skin cells need
to divide to replace damaged cells, but aged skin cells have
shorter telomeres and are less capable of dividing (Fossel,
REVERSING HUMAN AGING. William Morrow & Company, New York City,
1996).
[0080] By practicing the subject methods, e.g., via administration
of an active agent topically, one can extend telomere length, and
slow the downward spiral that skin experiences with age. Such a
product not only helps protect a person against the impairments of
aging skin; it also permits rejuvenated skin cells to restore
youthful immune resistance and appearance. The subject methods can
be used for both medical and cosmetic skin rejuvenation
applications.
[0081] Yet another disease condition in which the subject methods
find use in the treatment of osteoporosis. Two types of cells
interplay in osteoporosis: osteoblasts make bone and osteoclasts
destroy it. Normally, the two are in balance and maintain a
constant turnover of highly structured bone. In youth, bones are
resilient, harder to break, and heal quickly. In old age, bones are
brittle, break easily, and heal slowly and often improperly. Bone
loss has been postulated to occur because aged osteoblasts, having
lost much of their replicative capacity, cannot continue to divide
at the rate necessary to maintain balance (Hazzard et al.
PRINCIPLES OF GERIATRIC MEDICINE AND GERONTOLOGY, 2d ed.
McGraw-Hill, New York City, 1994). The subject methods can be
employed to lengthen telomeres of osteoblast and osteoclast stem
cells, thereby encouraging bone replacement and proper remodeling
and reinforcement. The resultant stronger bone improves the quality
of life for the many sufferers of osteoporosis and provides savings
from fewer fracture treatments. The subject methods are generally
part of a comprehensive treatment regime that also includes
calcium, estrogen, and exercise.
[0082] Additional disease conditions in which the subject methods
find use are described in WO 99/35243, the disclosures of which are
herein incorporated by reference.
[0083] In addition to the above described methods, the subject
methods can also be used to extend the lifetime of a mammal. By
extend the lifetime is meant to increase the time during which the
animal is alive, where the increase is generally at least 1 %,
usually at least 5% and more usually at least about 10%, as
compared to a control.
[0084] As indicated above, instead of a multicellular animal, the
target may be a cell or population of cells which are treated
according to the subject methods and then introduced into a
multicellular organism for therapeutic effect. For example, the
subject methods may be employed in bone marrow transplants for the
treatment of cancer and skin grafts for burn victims. In these
cases, cells are isolated from a human donor and then cultured for
transplantation back into human recipients. During the cell
culturing, the cells normally age and senesce, decreasing their
useful lifespans. Bone marrow cells, for instance, lose
approximately 40% of their replicative capacity during culturing.
This problem is aggravated when the cells are first genetically
engineered (Decary, Mouly et al. Hum Gene Ther 7(11): 1347-50,
1996). In such cases, the therapeutic cells must be expanded from a
single engineered cell. By the time there are sufficient cells for
transplantation, the cells have undergone the equivalent of 50
years of aging (Decary, Mouly et al. Hum Gene Ther 8(12): 1429-38,
1997). Use of the subject methods spares the replicative capacity
of bone marrow cells and skin cells during culturing and expansion
and thus significantly improves the survival and effectiveness of
bone marrow and skin cell transplants. Any transplantation
technology requiring cell culturing can benefit from the subject
methods, including ex vivo gene therapy applications in which cells
are cultured outside of the animal and then administered to the
animal, as described in U.S. Pat. Nos. 6,068,837; 6,027,488;
5,824,655; 5,821,235; 5,770,580; 5,756,283; 5,665,350; the
disclosures of which are herein incorporated by reference.
[0085] Treatment of Disease Conditions by Decreasing TERT
Expression
[0086] As summarized above, also provided are methods for enhancing
repression of TERT expression, where by enhancement of TERT
expression repression is meant a decrease in TERT expression by a
factor of at least about 2 fold, usually at least about 5 fold and
more usually at least about 10 fold, as compared to a control.
Methods for enhancing Site C mediated repression of TERT expression
find use in, among other applications, the treatment of cellular
proliferative disease conditions, particularly abnormal cellular
proliferative disease conditions, including, but not limited to,
neoplastic disease conditions, e.g., cancer. In such applications,
an effective amount of an active agent, e.g., a Site C repressor
protein, analog or mimetic thereof, a vector encoding a Site C
repressor protein or active fragment thereof, an agent that
enhances endogenous Site C repressor activity, an agent that
enhances expression of Site C repressor protein, etc., is
administered to the subject in need thereof. Treatment is used
broadly as defined above, e.g., to include at least an amelioration
in one or more of the symptoms of the disease, as well as a
complete cessation thereof, as well as a reversal and/or complete
removal of the disease condition, e.g., cure. Methods of treating
disease conditions resulting from unwanted TERT expression, such as
cancer and other diseases characterized by the presence of unwanted
cellular proliferation, are described in, for example, U.S. Pat.
Nos. 5,645,986; 5,656,638; 5,703,116; 5,760,062; 5,767,278;
5,770,613; and 5,863,936; the disclosures of which are herein
incorporated by reference.
[0087] Nucleic Acid Compositions
[0088] Also provided by the subject invention are nucleic acid
compositions, where the compositions are present in other than
their natural environment, e.g., are isolated, recombinant, etc.,
that include a Site C repressor binding site/domain/region, as
described above. In other embodiments, the subject nucleic acids
have a sequence that is substantially the same as, or identical to,
the Site C repressor binding site sequences as described above,
e.g., SEQ ID NOs: 01 to 04. A given sequence is considered to be
substantially similar to this particular sequence if it shares high
sequence similarity with the above described specific sequences,
e.g. at least 75% sequence identity, usually at least 90%, more
usually at least 95% sequence identity with the above specific
sequences. Sequence similarity is calculated based on a reference
sequence, which may be a subset of a larger sequence. A reference
sequence will usually be at least about 18 nt long, more usually at
least about 30 nt long, and may extend to the complete sequence
that is being compared. Algorithms for sequence analysis are known
in the art, such as BLAST, described in Altschul et al. (1990), J.
Mol. Biol. 215:403-10 (using default settings, i.e. parameters w=4
and T=17). Of particular interest in certain embodiments are
nucleic acids of substantially the same length as the specific
nucleic acid identified above, where by substantially the same
length is meant that any difference in length does not exceed about
20 number %, usually does not exceed about 10 number % and more
usually does not exceed about 5 number %; and have sequence
identity to this sequence of at least about 90%, usually at least
about 95% and more usually at least about 99% over the entire
length of the nucleic acid.
[0089] Also provided are nucleic acids that hybridize to the above
described nucleic acid under stringent conditions. An example of
stringent hybridization conditions is hybridization at 50.degree.
C. or higher and 0.1.times.SSC (15 mM sodium chloride/1.5 mM sodium
citrate). Another example of stringent hybridization conditions is
overnight incubation at 42.degree. C. in a solution: 50% formamide,
5.times.SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium
phosphate (pH7.6), 5.times. Denhardt's solution, 10% dextran
sulfate, and 20 .mu.g/ml denatured, sheared salmon sperm DNA,
followed by washing the filters in 0.1.times.SSC at about
65.degree. C. Stringent hybridization conditions are hybridization
conditions that are at least as stringent as the above
representative conditions, where conditions are considered to be at
least as stringent if they are at least about 80% as stringent,
typically at least about 90% as stringent as the above specific
stringent conditions. Other stringent hybridization conditions are
known in the art and may also be employed to identify nucleic acids
of this particular embodiment of the invention.
[0090] In many embodiments, the above described nucleic acid
compositions include the Site C sequence/domain region but do not
include the full sequence of the hTERT minimal promoter. In these
embodiments, the subject nucleic acids include no more than about
90 number %, usually no more than about 80 number % and more
usually no more than about 75 number %, where in many embodiments
the subject nucleic acids include less than about 50 number %,
sometimes less than about 40 number % and sometimes less than about
25 number % of the total sequence of the hTERT minimal promoter. In
certain embodiments, the length of the subject nucleic acids ranges
from about 5 to about 5000 bases, sometimes from about 10 to about
2500 bases and usually from about 10 to about 1000 bases, where in
certain embodiments the length ranges from about 10 to about 500
bases, sometimes from about 10 to about 250 bases and sometimes
from about 10 to about 100 bases, including from about 10 to about
50 bases.
[0091] The above described nucleic acid compositions find use in a
variety of different applications, including the preparation of
constructs, e.g., vectors, expression systems, etc., as described
more fully below, the preparation of probes for the Site C
repressor binding site sequence in non-human animals, i.e.,
non-human Site C repressor binding site homologs, and the like.
Where the subject nucleic acids are employed as probes, a fragment
of the provided nucleic acid may be used as a hybridization probe
against a genomic library from the target organism of interest,
where low stringency conditions are used. The probe may be a large
or small fragment, generally ranging in length from about 10 to 100
nt, usually from about 15 to 50 nt. Nucleic acids having sequence
similarity are detected by hybridization under low stringency
conditions, for example, at 50.degree. C. and 6.times.SSC (0.9 M
sodium chloride/0.09 M sodium citrate) and remain bound when
subjected to washing at 55.degree. C. in 1.times.SSC (0.15 M sodium
chloride/0.015 M sodium citrate). Sequence identity may be
determined by hybridization under stringent conditions, for
example, at 50.degree. C. or higher and 0.1.times.SSC (15 mM sodium
chloride/01.5 mM sodium citrate). Nucleic acids having a region of
substantial identity to the provided nucleic acid sequences bind to
the provided sequences under stringent hybridization conditions. By
using probes, particularly labeled probes of DNA sequences, one can
isolate homologous or related sequences.
[0092] The subject nucleic acids are isolated and obtained in
substantial purity, generally as other than an intact chromosome.
As such, they are present in other than their naturally occurring
environment. Usually, the DNA will be obtained substantially free
of other nucleic acid sequences that do not include a Site C
repressor binding site sequence or fragment thereof, generally
being at least about 50%, usually at least about 90% pure and are
typically "recombinant", i.e. flanked by one or more nucleotides
with which it is not normally associated on a naturally occurring
chromosome.
[0093] The subject nucleic acids may be produced using any
convenient protocol, including synthetic protocols, e.g., those
where the nucleic acid is synthesized by a sequential monomeric
approach (e.g., via phosphoramidite chemistry); where subparts of
the nucleic acid are so synthesized and then assembled or
concatamerized into the final nucleic acid, and the like. Where the
nucleic acid of interest has a sequence that occurs in nature, the
nucleic acid may be retrieved, isolated, amplified etc., from a
natural source using conventional molecular biology protocols.
[0094] Also provided are nucleic acid compositions that include a
modified or altered Site C site, e.g., where the site includes one
or more deletions or substitutions as compared to the above
specific Site C sequences, including a deletion or substitution of
all or portion of the Site C repressor binding site, e.g.,
preferably a deletion or substitution of at least one nucleotide,
in certain embodiments at least four nucleotides within the region
of nucleotides from about -40 to about -90, usually from about -45
to about -85 and more usually from about -45 to about -80 relative
to the "A" of the telomerase ATG codon, including the specific
regions specified above, and usually at least 7 nucleotides from
this region, and preferably all nucleotides from this region.
Additionally, such a deletion may extend further, for example to
include the nucleotides from positions -74 to -58, or subsets
thereof, with the exception being deletions that result in the
presence of a site which in fact binds to the Site C repressor
protein in a manner that enhances TERT expression. The subject
nucleic acids of this embodiment that include a deletion (or
substitution) in all or a portion of the Site C repressor site of
the TERT promoter may be present in the genome of a cell or animal
of interest, e.g., as a "knockout" deletion in a transgenic cell or
animal, where the cell or animal initially has this region, or may
be present in an isolated form. A "knockout" animal could be
produced from an animal that originally has the subject Site C
repressor site using the sequences flanking specific Site C regions
described here and the basic "knockout" technology known to those
skilled in the art e.g. see U.S. Pat. No. 5,464,764 to
Capecchi.
[0095] Also provided are constructs comprising the subject nucleic
acid compositions, e.g., those that include the Site C repressor
binding site or those that include a deletion in the Site C
repressor binding site, inserted into a vector, where such
constructs may be used for a number of different applications,
including propagation, screening, genome alteration, and the like,
as described in greater detail below. Constructs made up of viral
and non-viral vector sequences may be prepared and used, including
plasmids, as desired. The choice of vector will depend on the
particular application in which the nucleic acid is to be employed.
Certain vectors are useful for amplifying and making large amounts
of the desired DNA sequence. Other vectors are suitable for
expression in cells in culture, e.g., for use in screening assays.
Still other vectors are suitable for transfer and expression in
cells in a whole animal or person. The choice of appropriate vector
is well within the skill of the art. Many such vectors are
available commercially. To prepare the constructs, the partial or
full-length nucleic acid is inserted into a vector typically by
means of DNA ligase attachment to a cleaved restriction enzyme site
in the vector. Alternatively, the desired nucleotide sequence can
be inserted by homologous recombination in vivo. Typically this is
accomplished by attaching regions of homology to the vector on the
flanks of the desired nucleotide sequence. Regions of homology are
added by ligation of oligonucleotides, or by polymerase chain
reaction using primers comprising both the region of homology and a
portion of the desired nucleotide sequence, for example. Additional
examples of nucleic acid compositions that include the Site C
repressor binding site are polymers, e.g. a double stranded DNA
molecules, that mimic the Site C repressor site as described above.
Also of interest are anti-sense sequences which are sufficiently
homologous to the Site C binding site, such that they are useful to
block attachment of the repressor protein to the Site C repressor
binding site.
[0096] Also provided are expression cassettes, vectors or systems
that find use in, among other applications, screening for agents
that modulate, e.g., inhibit or enhance the repressive activity of
the region, as described in greater detail below; and/or to provide
for expression of proteins under the control of the expression
regulation mechanism of the TERT gene. By expression cassette or
system is meant a nucleic acid that includes a sequence encoding a
peptide or protein of interest, i.e., a coding sequence, operably
linked to a promoter sequence, where by operably linked is meant
that expression of the coding sequence is under the control of the
promoter sequence. The expression systems and cassettes of the
subject invention comprise a Site C repressor binding site/region
operably linked to the promoter, where the promoter is, in many
embodiments, a TERT promoter, such as the hTERT promoter. See e.g.,
the hTERT promoter sequence described in Cong et al., Hum. Mol.
Genet. (1999) 8:137-142.
[0097] As indicated above, expression systems comprising the
subject regions find use in applications where it is desired to
control expression of a particular coding sequence using the TERT
transcriptional mechanism. In such applications, the expression
system further includes the coding sequence of interest operably
linked to the TERT promoter/Site C repressor binding site elements.
The expression system is then employed in an appropriate
environment to provide expression or non-expression of the protein,
as desired, e.g., in an environment in which telomerase is
expressed, e.g., a Hela cell, or in an environment in which
telomerase is not expressed, e.g., an MRC5 cell. Alternatively, the
expression system may be used in an environment in which telomerase
expression is inducible, e.g., by adding to the system an
additional agent that turns on telomerase expression.
[0098] The above applications of the subject nucleic acid
compositions are merely representative of the diverse applications
in which the subject nucleic acid compositions find use.
[0099] Generation of Antibodies
[0100] Also provided are methods of generating antibodies, e.g.,
monoclonal antibodies. In one embodiment, the blocking or
inhibition, either directly or indirectly as described above, of
the Site C repressor site/Site C repressor interaction is used to
immortalize cells in culture, e.g., by enhancing telomerase
expression. Exemplary of cells that may be used for this purpose
are non-transformed antibody producing cells, e.g. B cells and
plasma cells which may be isolated and identified for their ability
to produce a desired antibody using known technology as, for
example, taught in U.S. Pat. No. 5,627,052. These cells may either
secrete antibodies (antibody-secreting cells) or maintain
antibodies on the surface of the cell without secretion into the
cellular environment. Such cells have a limited lifespan in
culture, and are usefully immortalized by upregulating expression
of telomerase using the methods of the present invention.
[0101] Because the above described methods are methods of
increasing expression of TERT and therefore increasing the
proliferative capacity and/or delaying the onset of senescence in a
cell, they find applications in the production of a range of
reagents, typically cellular or animal reagents. For example, the
subject methods may be employed to increase proliferation capacity,
delay senescence and/or extend the lifetimes of cultured cells.
Cultured cell populations having enhanced TERT expression are
produced using any of the protocols as described above, including
by contact with an agent that inhibits repressor region
transcription repression and/or modification of the repressor
region in a manner such that it no longer represses TERT coding
sequence transcription, etc.
[0102] The subject methods find use in the generation of monoclonal
antibodies. An antibody-forming cell may be identified among
antibody-forming cells obtained from an animal which has either
been immunized with a selected substance, or which has developed an
immune response to an antigen as a result of disease. Animals may
be immunized with a selected antigen using any of the techniques
well known in the art suitable for generating an immune response.
Antigens may include any substance to which an antibody may be
made, including, among others, proteins, carbohydrates, inorganic
or organic molecules, and transition state analogs that resemble
intermediates in an enzymatic process. Suitable antigens include,
among others, biologically active proteins, hormones, cytokines,
and their cell surface receptors, bacterial or parasitic cell
membrane or purified components thereof, and viral antigens.
[0103] As will be appreciated by one of ordinary skill in the art,
antigens which are of low immunogenicity may be accompanied with an
adjuvant or hapten in order to increase the immune response (for
example, complete or incomplete Freund's adjuvant) or with a
carrier such as keyhole limpet hemocyanin (KLH).
[0104] Procedures for immunizing animals are well known in the art.
Briefly, animals are injected with the selected antigen against
which it is desired to raise antibodies. The selected antigen may
be accompanied by an adjuvant or hapten, as discussed above, in
order to further increase the immune response. Usually the
substance is injected into the peritoneal cavity, beneath the skin,
or into the muscles or bloodstream. The injection is repeated at
varying intervals and the immune response is usually monitored by
detecting antibodies in the serum using an appropriate assay that
detects the properties of the desired antibody. Large numbers of
antibody-forming cells can be found in the spleen and lymph node of
the immunized animal. Thus, once an immune response has been
generated, the animal is sacrificed, the spleen and lymph nodes are
removed, and a single cell suspension is prepared using techniques
well known in the art.
[0105] Antibody-forming cells may also be obtained from a subject
which has generated the cells during the course of a selected
disease. For instance, antibody-forming cells from a human with a
disease of unknown cause, such as rheumatoid arthritis, may be
obtained and used in an effort to identify antibodies which have an
effect on the disease process or which may lead to identification
of an etiological agent or body component that is involved in the
cause of the disease. Similarly, antibody-forming cells may be
obtained from subjects with disease due to known etiological agents
such as malaria or AIDS. These antibody forming cells may be
derived from the blood or lymph nodes, as well as from other
diseased or normal tissues. Antibody-forming cells may be prepared
from blood collected with an anticoagulant such as heparin or EDTA.
The antibody-forming cells may be further separated from
erythrocytes and polymorphs using standard procedures such as
centrifugation with Ficoll-Hypaque (Pharmacia, Uppsula, Sweden).
Antibody-forming cells may also be prepared from solid tissues such
as lymph nodes or tumors by dissociation with enzymes such as
collagenase and trypsin in the presence of EDTA.
[0106] Antibody-forming cells may also be obtained by culture
techniques such as in vitro immunization. Briefly, a source of
antibody-forming cells, such as a suspension of spleen or lymph
node cells, or peripheral blood mononuclear cells are cultured in
medium such as RPMI 1640 with 10% fetal bovine serum and a source
of the substance against which it is desired to develop antibodies.
This medium may be additionally supplemented with amounts of
substances known to enhance antibody-forming cell activation and
proliferation such as lipopolysaccharide or its derivatives or
other bacterial adjuvants or cytokines such as IL-1, IL-2, IL-4,
IL-5, IL-6, GM-CSF, and IFN-.gamma. To enhance immunogenicity, the
selected antigen may be coupled to the surface of cells, for
example, spleen cells, by conventional techniques such as the use
of biotin/avidin as described below.
[0107] Antibody-forming cells may be enriched by methods based upon
the size or density of the antibody-forming cells relative to other
cells. Gradients of varying density of solutions of bovine serum
albumin can also be used to separate cells according to density.
The fraction that is most enriched for desired antibody-forming
cells can be determined in a preliminary procedure using the
appropriate indicator system in order to establish the
antibody-forming cells.
[0108] The identification and culture of antibody producing cells
of interest is followed by enhancement of TERT expression in these
cells by the subject methods, thereby avoiding the need for the
immortalization/fusing step employed in traditional hybridoma
manufacture protocols. In such methods, the first step is
immunization of the host animal with an immunogen, typically a
polypeptide, where the polypeptide will preferably be in
substantially pure form, comprising less than about 1% contaminant.
The immunogen may comprise the complete protein, fragments or
derivatives thereof. To increase the immune response of the host
animal, the protein may be combined with an adjuvant, where
suitable adjuvants include alum, dextran sulfate, large polymeric
anions, oil & water emulsions, e.g. Freund's adjuvant, Freund's
complete adjuvant, and the like. The protein may also be conjugated
to synthetic carrier proteins or synthetic antigens. A variety of
hosts may be immunized to produce the subject antibodies. Such
hosts include rabbits, guinea pigs, rodents (e.g. mice, rats),
sheep, goats, and the like. The protein is administered to the
host, usually intradermally, with an initial dosage followed by one
or more, usually at least two, additional booster dosages.
Following immunization, generally, the spleen and/or lymph nodes of
an immunized host animal provide a source of plasma cells. The
plasma cells are treated according to the subject invention to
enhance TERT expression and thereby, increase the proliferative
capacity and/or delay senescence to produce "pseudo" immortalized
cells. Culture supernatant from individual cells is then screened
using standard techniques to identify those producing antibodies
with the desired specificity. Suitable animals for production of
monoclonal antibodies to a human protein include mouse, rat,
hamster, etc. To raise antibodies against the mouse protein, the
animal will generally be a hamster, guinea pig, rabbit, etc. The
antibody may be purified from the cell supernatants or ascites
fluid by conventional techniques, e.g. affinity chromatography
using RFLAT-1protein bound to an insoluble support, protein A
sepharose, etc.
[0109] In an analogous fashion, the subject methods are employed to
enhance TERT expression in non-human animals, e.g., non-human
animals employed in laboratory research. Using the subject methods
with such animals can provide a number of advantages, including
extending the lifetime of difficult and/or expensive to produce
transgenic animals. As with the above described cells and cultures
thereof, the expression of TERT in the target animals may be
enhanced using a number of different protocols, including the
administration of an agent that inhibits Site C repressor protein
repression and/or targeted disruption of the Site C repressor
binding site. The subject methods may be used with a number of
different types of animals, where animals of particular interest
include mammals, e.g., rodents such as mice and rats, cats, dogs,
sheep, rabbits, pigs, cows, horses, and non-human primates, e.g.
monkeys, baboons, etc.
[0110] Screening Assays
[0111] Also provided by the subject invention are screening
protocols and assays for identifying agents that modulate, e.g.,
inhibit or enhance, Site C repression of TERT transcription. The
screening methods include assays that provide for
qualitative/quantitative measurements of TERT promoter controlled
expression, e.g., of a coding sequence for a marker or reporter
gene, in the presence of a particular candidate therapeutic agent.
Assays of interest include assays that measures the TERT promoter
controlled expression of a reporter gene (i.e. coding sequence,
e.g., luciferase, SEAP, etc.) in the presence and absence of a
candidate inhibitor agent, e.g., the expression of the reporter
gene in the presence or absence of a candidate agent. The screening
method may be an in vitro or in vivo format, where both formats are
readily developed by those of skill in the art. Whether the format
is in vivo or in vitro, an expression system, e.g., a plasmid, that
includes a Site C repressor binding site, a TERT promoter and a
reporter coding sequence all operably linked is combined with the
candidate agent in an environment in which, in the absence of the
candidate agent, the TERT promoter is repressed, e.g., in the
presence of the Site C repressor protein that interacts with the
Site C repressor binding site and causes TERT promoter repression.
The conditions may be set up in vitro by combining the various
required components in an aqueous medium, or the assay may be
carried out in vivo, e.g., in a cell that normally lacks telomerase
activity, e.g., an MRC5 cell, etc.
[0112] A variety of different candidate agents may be screened by
the above methods. Candidate agents encompass numerous chemical
classes, though typically they are organic molecules, preferably
small organic compounds having a molecular weight of more than 50
and less than about 2,500 daltons. Candidate agents comprise
functional groups necessary for structural interaction with
proteins, particularly hydrogen bonding, and typically include at
least an amine, carbonyl, hydroxyl or carboxyl group, preferably at
least two of the functional chemical groups. The candidate agents
often comprise cyclical carbon or heterocyclic structures and/or
aromatic or polyaromatic structures substituted with one or more of
the above functional groups. Candidate agents are also found among
biomolecules including peptides, saccharides, fatty acids,
steroids, purines, pyrimidines, derivatives, structural analogs or
combinations thereof.
[0113] Candidate agents are obtained from a wide variety of sources
including libraries of synthetic or natural compounds. For example,
numerous means are available for random and directed synthesis of a
wide variety of organic compounds and biomolecules, including
expression of randomized oligonucleotides and oligopeptides.
Alternatively, libraries of natural compounds in the form of
bacterial, fungal, plant and animal extracts are available or
readily produced. Additionally, natural or synthetically produced
libraries and compounds are readily modified through conventional
chemical, physical and biochemical means, and may be used to
produce combinatorial libraries. Known pharmacological agents may
be subjected to directed or random chemical modifications, such as
acylation, alkylation, esterification, amidification, etc. to
produce structural analogs.
[0114] Agents identified in the above screening assays that inhibit
Site C repression of TERT transcription find use in the methods
described above, e.g., in the enhancement of TERT expression.
Alternatively, agents identified in the above screening assays that
enhance Site C repression find use in applications where inhibition
of TERT expression is desired, e.g., in the treatment of disease
conditions characterized by the presence of unwanted TERT
expression, such as cancer and other diseases characterized by the
presence of unwanted cellular proliferation, where such methods are
described in, for example, U.S. Pat. Nos. 5,645,986; 5,656,638;
5,703,116; 5,760,062; 5,767,278; 5,770,613; and 5,863,936; the
disclosures of which are herein incorporated by reference.
[0115] The following examples are offered by way of illustration
and not by way of limitation.
Experimental
[0116] I. Deletion Experiments
[0117] 118 deletions of the minimal telomerase promoter as shown in
FIG. 1 were constructed (using site specific in vitro mutagenesis
as described in U.S. Pat. No. 5,702,931, the disclosure of which is
herein incorporated by reference) to find regions within the
telomerase promoter that contain potential repressor sites. These
deletions ranged in size from 10 to 300 bases. Each deletion
version of the minimal promoter was tested for its ability to
express SEAP in MRC5 and HELA. Several of the deletions, all
mapping about 50-100 bases upstream of the telomerase translation
initiation codon (ATG), showed .about.10 fold increased expression.
The region was called the Site C region. The highest expression in
MRC5 was obtained with the deletion called 11K. This 30 base
deletion includes bases -48 to -77 relative to the translation
initiation codon ATG. However, a similar deletion, called 12K, that
includes bases -48 to -57 results in 500 fold less expression. On
the other hand, when 11K and 12K were compared in HELA, they both
gave equivalent amounts of expression. The repressor site in the
Site C region therefore is contained, or overlaps with, the 20
bases present in 12K and absent in 11K (i.e. -58 to -77).
[0118] To identify more specifically the bases that make up this
repressor site, additional deletions were made. Each deletion is 10
bases long with 7 to 8 base overlaps between consecutive deletions.
The deletions were made in the minimal telomerase promoter in the
plasmid designated pSSI20 (the full annotated sequence of pSSI20 is
provided in FIG. 2). Each deletion mutant was independently made
three times and all deletions were transiently transfected into
MRC5 (telomerase negative normal cells) and HELA (telomerase
positive immortal cells).
[0119] A portion of the 5' untranslated region is shown below, from
-77 to 1, the start of translation. The repressor site extends from
-77 to -48, as shown.
7 CTCCTCGC GGCGCGAGTT TCAGGCAGCG (SEQ ID NO.: 7) CTGCGTCCTG
CTGCGCACGT GGGAAGCCCT repressor site (-77 to -48) GGCCCCGGCC
ACCCCCGCGA .vertline. start codon (1)
[0120] Of particular interest are sequences with a deletion
extending from -67 to -58, comprising the nucleotides CGCGAGTTTC
(SEQ ID NO:03).
[0121] The expression levels were measured using the Secreted
Alkaline Phosphatase Assay (SEAP Assay) commercially available from
Clontech (Palo Alto, Calif.). The results are shown below.
8 Deletion MRC5 HELA NONE (control) 0.1931 78.3076 -104 to -95 0.19
78.30 -102 to -93 4.92 73.97 -99 to -90 1.19 86.95 -97 to -88 1.69
97.94 -94 to -85 8.06 89.6 -92 to -83 7.89 89.86 -89 to -80 12.00
93.91 -87 to -78 7.26 59.74 -84 to -75 7.77 85.48 -82 to -73 4.83
99.4 -79 to -70 3.79 73.34 -77 to -68 17.15 82.26 -74 to -65 34.44
78.99 -72 to -63 33.22 123.8 -69 to -60 33.15 133.56 -67 to -58
56.98 97.74 -64 to -55 21.82 127.32 -62 to -53 4.60 108 -59 to -50
19.58 103.1
[0122] The column of deletions indicates the bases that were
deleted in the repressor site, which is indicated relative to the
AUG start codon. The columns for MRC5 and HELA show the level of
expression observed for each deletion, reported as a percentage of
the SV40 early promoter, which was used to normalize the two cell
lines.
[0123] The data demonstrate that the deletion from "-67 to -58"
gave a reading of 56.9852, as compared to a reading of 0.193109 in
the control cells with no deletion in the promoter, giving an
increase of 295 fold higher expression. This same deletion gave
only 97.746 in HELA cells, compared to the undeleted control value
of 78.3076, resulting in a 1.25 fold higher expression. This
indicates that a repressor function operates in MRC5 cells to
repress expression of the wild type telomerase promoter. When the
expression level of deletion "-67 to -58" in MRC5 is compared to
the wild type promoter in HELA it is observed that the deletion
resulted in almost as much expression as the levels observed in
HELA that are sufficient to maintain telomere length. That is, the
expression of the deletion in MRC5 was 59.9852/78.3076=77% of the
wild type in HELA. This indicates that depressing the repressor in
MRC5 allows for sufficient amounts of telomerase expression to
maintain the length of the telomeres in the cells during cell
division, and to stop cellular aging in these cells.
[0124] II. Identification of E2F Consensus Sequence
[0125] The Site C region described above was analyzed for the
presence of consensus sequences and an E2F transcription factor
binding site consensus sequence (E2F-Q6) was identified (see below)
utilizing software and databases provided by Genomatix
(http://genomatix.gsf.de). This identified consensus sequence is
located at -68 to -58 of the TERT promoter, or:
9 CTCCTCGC GGCGCGAGTT TCAGGCAGCG (SEQ ID NO.: 7) CTGCGTCCTG
CTGCGCACGT GGGAAGCCCT repressor site (-68 to -58) GGCCCCGGCC
ACCCCCGCGA .vertline. start codon (1)
[0126] The identified consensus sequence, E2F-Q6, includes all of
above described -67 to -58 deletion plus one base upstream. As can
be seen from the above results, every deletion that overlaps this
-67 to -58 site causes an elevation in expression with maximum
expression occurring from the deletion of bases -67 to -58. The
only exception to this general rule is the deletion from -62 to
-53. This deletion actually, accidentally, creates a new sequence
that matches the consensus E2F-Q6 sequence better than the original
-67 to -58 site does.
[0127] III. Fine Mapping of the Site C Site
[0128] A "fine mapping" analysis of the Site C binding site was
completed to determine the effect of each base within site C on
telomerase repression and the results are tabulated below and shown
graphically in FIG. 3. The "fine mapping" analysis involved single
base mutations or deletions within Site C and assayed for their
affects on the TERT promoter's ability to drive the expression of
the SEAP reporter gene in transient transfection assays. In the
graph of FIG. 3 the letters on the X-axis labeled "before" are the
bases of Site C before mutagenesis. The letters labeled "after" are
what the bases were changed to by in vitro mutagenesis. In this
experiment only one base was changed at a time. That is, in one
plasmid the C at -70 was changed to an A. That was the only change
that took place in the plasmid. In another plasmid A at -63 was
changed to a T. Again, that was the only change that took place in
the plasmid. Each plasmid was then transiently transfected into
MRC5 cells and expression of SEAP was assayed. The first data point
shows the expression of SEAP under control of the wild type
telomerase minimal promoter. This shows almost zero (83.10 SEAP
units) expression. The next data point shows SEAP expression when
the entire 10 base Site C sequence (SEQ ID NO. 03) is deleted. All
the subsequent data points show the expression resulting from each
of the single base changes shown in the X-axis.
[0129] This analysis resulted in the identification of the specific
bases within site C that control the regulation of the telomerase
promoter. Bases within the site C repressor binding site which were
found to be influential in telomerase repression are shown in the
site C sequence below as capital letters while those bases when
mutated or deleted had little or no effect on telomerase repression
are shown in small case.
[0130] Site C "fine mapping" results--CGCGagtTTc SEQ ID NO. 08
[0131] These results also show that the sequence that the Site C
binding protein binds to is GGCGCGAGTTTCA (SEQ ID NO:02).
10 Plasmid Base # Mutation SEAP pSSI20 Wild Type 83.10 pSSI304 -67
to -58 deleted 3093.70 pSSI658 -72 G->C 268.37 pSSI663 -71
A->G 208.63 pSSI664 -70 A->C 256.93 pSSI667 -69 C->G
596.70 pSSI552 -68 C->G 879.20 pSSI645 -67 G->C 1841.70
pSSI670 -66 C->G 3021.37 pSSI673 -65 A->C 3274.37 pSSI677 -64
A->G 2115.03 pSSI679 -63 T->A 968.70 pSSI682 -62 C->G
542.80 pSSI686 -61 C->T 1286.37 pSSI688 -60 C->T 2032.37
pSSI691 -59 A->T 2005.03 pSSI694 -58 A->C 1328.70 pSSI697 -57
T->A 1047.03 pSSI700 -56 A->G 66.27 pSSI703 -55 A->G
185.03 pSSI706 -54 G->C 369.03 pSSI710 -53 G->A 237.70
[0132] IV. Gel Shift Characterization of the Site C Site
[0133] The following oligos (each one was made double stranded)
were employed in gel shift experiments using nuclear extracts from
the normal cell line IMR90 and the immortal cell line Raji (Nuclear
extracts of IMR90, Catalog #1012217, and Raji, Catalog #100156 were
purchased from Geneka Biotechnology Inc.). IMR90 cells can be
obtained from ATCC Catalog #CCL-186. Raji cells can be obtained
from ATCC Catalog #CCL-86.). The gel shift protocol that was
followed is from the BandShift Kit, Amersham Pharmacia Biotech,
XY-026-00-06. The following changes to their protocol were made:
the binding reaction is at 4 C for 1 hour and no loading dye is
added prior to electrophoresis. The results were identical for
IMR90 and Raji. The ability of each oligo to shift in a gel shift
assay, using either nuclear extract, when radioactively labeled are
shown below.
11 Oligo Name Result Sequence SSI586 No Shift
TCTCCTCGCGGCGCGAGTTTCAGG (SEQ ID NO:09) SSI584 No Shift
TCTCCTCGCGGCGCGAGTTTCAGGCA (SEQ ID NO:10) SSI582 No Shift
TCTCCTCGCGGCGCGAGTTTCAGGCAGC (SEQ ID NO:11) SSI614 Weak Shift
TCTCCTCGCGGCGCGAGTTTCAGGCAGCG (SEQ ID NO:12) SSI570 Strong Shift
TCTCCTCGCGGCGCGAGTTTCAGGCAGCGC (SEQ ID NO:13) SSI630 Strong Shift
CTCGCGGCGCGAGTTTCAGGCAGCGC- TG (SEQ ID NO:14) SSI570 Strong Shift
TCTCCTCGCGGCGCGAGTTTCAGGCAGCGC (SEQ ID NO:15) SSI572 Strong Shift
TCCTCGCGGCGCGAGTTTCAGGCAGCGC (SEQ ID NO:16) SSI574 Strong Shift
CTCGCGGCGCGAGTTTCAGGCAGCGC (SEQ ID NO:17) SSI634 Strong Shift
CGCGGCGCGAGTTTCAGGCAGCGCTGCGTC (SEQ ID NO:18) SSI636 Strong Shift
CGGCGCGAGTTTCAGGCAGCGCTGCGTC (SEQ ID NO:19) SSI638 Weak Shift
GCGCGAGTTTCAGGCAGCGCTGCGTC (SEQ ID NO:20) SSI640 Weak Shift
CGCGAGTTTCAGGCAGCGCTGCGTC (SEQ ID NO:21) SSI642 Weak Shift
GCGAGTTTCAGGCAGCGCTGCGTC (SEQ ID NO:22)
[0134] The following mutant gel shift oligo (double stranded) in
which base -65 (relative to the ATG of telomerase) was converted
from a C to an A (shown in green) was also assayed.
12 TCTCCTCGCGGCGAGAGTTTCAGGCAGCGC (SEQ ID NO:23)
[0135] Previous SEAP assays (See Experiment "III. Fine Mapping of
the Site C Site" above) had shown that this mutation abolished
repressor binding as much as the complete deletion of the original
10 base site C sequence (SEQ ID NO:03). This mutant oligo did not
cause a shift, indicating that the gel shift data agrees with the
expression data.
[0136] As such, the following sequence is another Site C
sequence:
13 GGCGCGAGTTTCAGGCAGCGC (SEQ ID NO:04)
[0137] It is evident from the above results and discussion that the
subject invention provides important new nucleic acid compositions
that find use in a variety of applications, including the
establishment of expression systems that exploit the regulatory
mechanism of the TERT gene and the establishment of screening
assays for agents that enhance TERT expression. In addition, the
subject invention provides methods of enhancing TERT expression in
a cellular or animal host, which methods find use in a variety of
applications, including the production of scientific research
reagents and therapeutic treatment applications. Accordingly, the
subject invention represents significant contribution to the
art.
[0138] All publications and patents cited in this specification are
herein incorporated by reference as if each individual publication
or patent were specifically and individually indicated to be
incorporated by reference. The citation of any publication is for
its disclosure prior to the filing date and should not be construed
as an admission that the present invention is not entitled to
antedate such publication by virtue of prior invention.
[0139] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it is readily apparent to those of ordinary skill
in the art in light of the teachings of this invention that certain
changes and modifications may be made thereto without departing
from the spirit or scope of the appended claims.
Sequence CWU 1
1
25 1 41 DNA human 1 ggccccgccc tctcctcgcg gcgcgagttt caggcagcgc t
41 2 13 DNA human 2 ggcgcgagtt tca 13 3 10 DNA human 3 cgcgagtttc
10 4 21 DNA human 4 ggcgcgagtt tcaggcagcg c 21 5 85 PRT human 5 Gly
Arg Gly Arg His Pro Gly Lys Gly Val Lys Ser Pro Gly Glu Lys 1 5 10
15 Ser Arg Tyr Glu Thr Ser Leu Asn Leu Thr Thr Lys Arg Phe Leu Glu
20 25 30 Leu Leu Ser His Ser Ala Asp Gly Val Val Asp Leu Asn Trp
Ala Ala 35 40 45 Glu Val Leu Lys Val Gln Lys Arg Arg Ile Tyr Asp
Ile Thr Asn Val 50 55 60 Leu Glu Gly Ile Gln Leu Ile Ala Lys Lys
Ser Lys Asn His Ile Gln 65 70 75 80 Trp Leu Gly Ser His 85 6 76 PRT
human 6 Pro Pro Gly Thr Pro Ser Arg His Glu Lys Ser Leu Gly Leu Leu
Thr 1 5 10 15 Thr Lys Phe Val Ser Leu Leu Gln Glu Ala Lys Asp Gly
Val Leu Asp 20 25 30 Leu Lys Leu Ala Ala Asp Thr Leu Ala Val Arg
Gln Lys Arg Arg Ile 35 40 45 Tyr Asp Ile Thr Asn Val Leu Glu Gly
Ile Gly Leu Ile Glu Lys Lys 50 55 60 Ser Lys Asn Ser Ile Gln Trp
Lys Gly Val Gly Pro 65 70 75 7 78 DNA human 7 ctcctcgcgg cgcgagtttc
aggcagcgct gcgtcctgct gcgcacgtgg gaagccctgg 60 ccccggccac ccccgcga
78 8 10 DNA human 8 cgcgagtttc 10 9 24 DNA Artificial Sequence
synthetic oligonucleotide 9 tctcctcgcg gcgcgagttt cagg 24 10 26 DNA
Artificial Sequence synthetic oligonucleotide 10 tctcctcgcg
gcgcgagttt caggca 26 11 28 DNA Artificial Sequence synthetic
oligonucleotide 11 tctcctcgcg gcgcgagttt caggcagc 28 12 29 DNA
Artificial Sequence synthetic oligonucleotide 12 tctcctcgcg
gcgcgagttt caggcagcg 29 13 30 DNA Artificial Sequence synthetic
oligonucleotide 13 tctcctcgcg gcgcgagttt caggcagcgc 30 14 28 DNA
Artificial Sequence synthetic oligonucleotide 14 ctcgcggcgc
gagtttcagg cagcgctg 28 15 30 DNA Artificial Sequence synthetic
oligonucleotide 15 tctcctcgcg gcgcgagttt caggcagcgc 30 16 28 DNA
Artificial Sequence synthetic oligonucleotide 16 tcctcgcggc
gcgagtttca ggcagcgc 28 17 26 DNA Artificial Sequence synthetic
oligonucleotide 17 ctcgcggcgc gagtttcagg cagcgc 26 18 30 DNA
Artificial Sequence synthetic oligonucleotide 18 cgcggcgcga
gtttcaggca gcgctgcgtc 30 19 28 DNA Artificial Sequence synthetic
oligonucleotide 19 cggcgcgagt ttcaggcagc gctgcgtc 28 20 26 DNA
Artificial Sequence synthetic oligonucleotide 20 gcgcgagttt
caggcagcgc tgcgtc 26 21 25 DNA Artificial Sequence synthetic
oligonucleotide 21 cgcgagtttc aggcagcgct gcgtc 25 22 24 DNA
Artificial Sequence synthetic oligonucleotide 22 gcgagtttca
ggcagcgctg cgtc 24 23 30 DNA Artificial Sequence synthetic
oligonucleotide 23 tctcctcgcg gcgagagttt caggcagcgc 30 24 261 DNA
human 24 ccaggaccgc gctccccacg tggcggaggg actggggacc cgggcacccg
tcctgcccct 60 tcaccttcca gctccgcctc ctccgcgcgg accccgcccc
gtcccgaccc ctcccgggtc 120 cccggcccag ccccctccgg gccctcccag
cccctcccct tcctttccgc ggccccgccc 180 tctcctcgcg gcgcgagttt
caggcagcgc tgcgtcctgc tgcgcacgtg ggaagccctg 240 gccccggcca
cccccgcgat g 261 25 5928 DNA Artificial Sequence synthetic
expression plasmid 25 ggtaccgagc tcttacgcgt gctagcccgg gctcgagcca
ggaccgcgct ccccacgtgg 60 cggagggact ggggacccgg gcacccgtcc
tgccccttca ccttccagct ccgcctcctc 120 cgcgcggacc ccgccccgtc
ccgacccctc ccgggtcccc ggcccagccc cctccgggcc 180 ctcccagccc
ctccccttcc tttccgcggc cccgccctct cctcgcggcg cgagtttcag 240
gcagcgctgc gtcctgctgc gcacgtggga agccctggcc ccggccaccc ccgcgaattc
300 gcccaccatg ctgctgctgc tgctgctgct gggcctgagg ctacagctct
ccctgggcat 360 catcccagtt gaggaggaga acccggactt ctggaaccgc
gaggcagccg aggccctggg 420 tgccgccaag aagctgcagc ctgcacagac
agccgccaag aacctcatca tcttcctggg 480 cgatgggatg ggggtgtcta
cggtgacagc tgccaggatc ctaaaagggc agaagaagga 540 caaactgggg
cctgagatac ccctggccat ggaccgcttc ccatatgtgg ctctgtccaa 600
gacatacaat gtagacaaac atgtgccaga cagtggagcc acagccacgg cctacctgtg
660 cggggtcaag ggcaacttcc agaccattgg cttgagtgca gccgcccgct
ttaaccagtg 720 caacacgaca cgcggcaacg aggtcatctc cgtgatgaat
cgggccaaga aagcagggaa 780 gtcagtggga gtggtaacca ccacacgagt
gcagcacgcc tcgccagccg gcacctacgc 840 ccacacggtg aaccgcaact
ggtactcgga cgccgacgtg cctgcctcgg cccgccagga 900 ggggtgccag
gacatcgcta cgcagctcat ctccaacatg gacattgacg tgatcctagg 960
tggaggccga aagtacatgt ttcgcatggg aaccccagac cctgagtacc cagatgacta
1020 cagccaaggt gggaccaggc tggacgggaa gaatctggtg caggaatggc
tggcgaagcg 1080 ccagggtgcc cggtatgtgt ggaaccgcac tgagctcatg
caggcttccc tggacccgtc 1140 tgtgacccat ctcatgggtc tctttgagcc
tggagacatg aaatacgaga tccaccgaga 1200 ctccacactg gacccctccc
tgatggagat gacagaggct gccctgcgcc tgctgagcag 1260 gaacccccgc
ggcttcttcc tcttcgtgga gggtggtcgc atcgaccatg gtcatcatga 1320
aagcagggct taccgggcac tgactgagac gatcatgttc gacgacgcca ttgagagggc
1380 gggccagctc accagcgagg aggacacgct gagcctcgtc actgccgacc
actcccacgt 1440 cttctccttc ggaggctacc ccctgcgagg gagctccatc
ttcgggctgg cccctggcaa 1500 ggcccgggac aggaaggcct acacggtcct
cctatacgga aacggtccag gctatgtgct 1560 caaggacggc gcccggccgg
atgttaccga gagcgagagc gggagccccg agtatcggca 1620 gcagtcagca
gtgcccctgg acgaagagac ccacgcaggc gaggacgtgg cggtgttcgc 1680
gcgcggcccg caggcgcacc tggttcacgg cgtgcaggag cagaccttca tagcgcacgt
1740 catggccttc gccgcctgcc tggagcccta caccgcctgc gacctggcgc
cccccgccgg 1800 caccaccgac gccgcgcacc cgggttactc tagagtcggg
gcggccggcc gcttcgagca 1860 gacatgataa gatacattga tgagtttgga
caaaccacaa ctagaatgca gtgaaaaaaa 1920 tgctttattt gtgaaatttg
tgatgctatt gctttatttg taaccattat aagctgcaat 1980 aaacaagtta
acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg 2040
gaggtttttt aaagcaagta aaacctctac aaatgtggta aaatcgataa ggatccgtcg
2100 accgatgccc ttgagagcct tcaacccagt cagctccttc cggtgggcgc
ggggcatgac 2160 tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa
ctcgtaggac aggtgccggc 2220 agcgctcttc cgcttcctcg ctcactgact
cgctgcgctc ggtcgttcgg ctgcggcgag 2280 cggtatcagc tcactcaaag
gcggtaatac ggttatccac agaatcaggg gataacgcag 2340 gaaagaacat
gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 2400
tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
2460 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
ggaagctccc 2520 tcgtgcgctc tcctgttccg accctgccgc ttaccggata
cctgtccgcc tttctccctt 2580 cgggaagcgt ggcgctttct catagctcac
gctgtaggta tctcagttcg gtgtaggtcg 2640 ttcgctccaa gctgggctgt
gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 2700 ccggtaacta
tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 2760
ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
2820 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct
ctgctgaagc 2880 cagttacctt cggaaaaaga gttggtagct cttgatccgg
caaacaaacc accgctggta 2940 gcggtggttt ttttgtttgc aagcagcaga
ttacgcgcag aaaaaaagga tctcaagaag 3000 atcctttgat cttttctacg
gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 3060 ttttggtcat
gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 3120
gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
3180 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
gcctgactcc 3240 ccgtcgtgta gataactacg atacgggagg gcttaccatc
tggccccagt gctgcaatga 3300 taccgcgaga cccacgctca ccggctccag
atttatcagc aataaaccag ccagccggaa 3360 gggccgagcg cagaagtggt
cctgcaactt tatccgcctc catccagtct attaattgtt 3420 gccgggaagc
tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg 3480
ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
3540 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
agctccttcg 3600 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
atcactcatg gttatggcag 3660 cactgcataa ttctcttact gtcatgccat
ccgtaagatg cttttctgtg actggtgagt 3720 actcaaccaa gtcattctga
gaatagtgta tgcggcgacc gagttgctct tgcccggcgt 3780 caatacggga
taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac 3840
gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
3900 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
catgagcaaa 3960 aacaggaagg caaaatgccg caaaaaaggg aataagggcg
acacggaaat gttgaatact 4020 catactcttc ctttttcaat attattgaag
catttatcag ggttattgtc tcatgagcgg 4080 atacatattt gaatgtattt
agaaaaataa acaaataggg gttccgcgca catttccccg 4140 aaaagtgcca
cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag 4200
gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca
4260 catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga
gcagacaagc 4320 ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc
tggcttaact atgcggcatc 4380 agagcagatt gtactgagag tgcaccatat
gcggtgtgaa tctcgtacgc acgtgcctca 4440 gtacgtaaga ggttccaact
ttcaccataa tgaaataaga tcactaccgg gcgtattttt 4500 tgagttatcg
agattttcag gagctaagga agctaaaatg gagaaaaaaa tcactggata 4560
taccaccgtt gatatatccc aatggcatcg taaagaacat tttgaggcat ttcagtcagt
4620 tgctcaatgt acctataacc agaccgttca actggatatt acggcctttt
taaagaccgt 4680 aaagaaaaat aagcacaagt tttatccggc ctttattcac
attcttgccc gcctgatgaa 4740 tgctcatccc gagttccgta tggcaatgaa
agacggtgag ctggtgatat gggatagtgt 4800 tcacccttgt tacaccgttt
tccatgagca aactgaaacg ttttcatcgc tctggagtga 4860 ataccacgac
gatttccggc agtttctaca catatattcg caagatgtgg cgtgttacgg 4920
tgaaaacctg gcctatttcc ctaaagggtt tattgagaat atgtttttcg tctcagccaa
4980 tccctgggtg agtttcacca gttttgattt aaacgtggcc aatatggaca
acttcttcgc 5040 ccccgttttc acaatgggca aatattatac gcaaggcgac
aaggtgctga tgccgctggc 5100 gattcaggtt catcatgccg tttgtgatgg
cttccatgtc ggcagaatgc ttaatgaatt 5160 acaacagtgt accgcatcag
gcgaaattgt aaacgttaat attttgttaa aattcgcgta 5220 aatatttgtt
aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat 5280
aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
5340 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
gggcgatggc 5400 ccactacgtg aaccatcacc caaatcaagt tttttgcggt
cgaggtgccg taaagctcta 5460 aatcggaacc ctaaagggag cccccgattt
agagcttgac ggggaaagcc ggcgaacgtg 5520 gcgagaaagg aagggaagaa
agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg 5580 gtcacgctgc
gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc 5640
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
5700 tacgccagcc caagctacca tgataagtaa gtaatattaa ggtacgggag
gtacttggag 5760 cggccgcaat aaaatatctt tattttcatt acatctgtgt
gttggttttt tgtgtgaatc 5820 gatagtacta acatacgctc tccatcaaaa
caaaacgaaa caaaacaaac tagcaaaata 5880 ggctgtcccc agtgcaagtg
caggtgccag aacatttctc tatcgata 5928
* * * * *
References