Note: Descriptions are shown in the official language in which they were submitted.
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BRCA-1 REGULATORS AND METHODS OF USE
FIELD OF THE INVENTION
This invention relates generally to proliferative diseases such as cancer and,
more specifically, to regulation of the tumor suppressor BRCA-1 that can be
used to
diagnose and treat proliferative diseases.
BACKGROUND OF THE INVENTION
Cancer is one of the leading causes of death in the United States. Each year,
more than half a million Americans die from cancer, and more than one million
are
newly diagnosed with the disease. Cancerous tumors result when a cell escapes
from
its normal growth regulatory mechanisms and proliferates in an uncontrolled
fashion.
Tumor cells can metastasize to secondary sites if treatment of the primary
tumor is
either not complete or not initiated before substantial progression of the
disease.
Early diagnosis and effective treatment of tumors is, therefore, essential for
survival.
Cancer involves the clonal replication of populations of cells that have
gained
competitive advantage over.normal cells through the alteration of regulatory
genes.
Regulatory genes can be broadly classified into "oncogenes" which, when
activated
or overexpressed, promote unregulated cell proliferation, and "tumor
suppressor
genes," which when inactivated or underexpressed, fail to prevent abnormal
cell
proliferation. Loss of function or inactivation of tumor suppressor genes is
thought
to play a central role in the initiation and progression of a significant
number of
human cancers.
Mutations in one known tumor suppressor gene, BRCA-1, contribute in
essentially all cases to inherited susceptibility to ovarian and breast
cancers.
Additionally, BRCA-1 expression levels are reduced or undetectable in the
tumor
cells of sporadic breast cancers.
Approaches for treating cancer by modulating the function of tumor
suppressor genes, either with pharmaceutical compounds or by gene therapy
methods, have yielded promising results in animal models and in human clinical
trials. Approaches for diagnosing and prognosing cancer by identifying
mutations in
known tumor suppressor genes or mutations in genes that regulate the
expression of
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tumor suppressor genes have also been developed. For example, identifying
individuals containing germline mutations in known tumor suppressor genes has
permitted the identification of individuals at increased risk of developing
cancer.
Such individuals are then closely monitored or treated prophylactically to
improve
their chance of survival. Identifying the pattern of alterations of known
tumor
suppressor genes in biopsy samples is also being used to determine the
presence or
stage of a tumor. Being able to determine whether a cancer is benign or
malignant,
at an early or late stage of progression, provides the patient and clinician
with a more
accurate prognosis and can be used to determine the most effective treatment
for the
patient.
In view of the importance of tumor suppressor molecules in the detection and
treatment of cancer, and the known correlation of the tumor suppressor BRCA-1
with breast and ovarian cancers, there exists a need to identify nucleic acids
and
polypeptides that influence the level or activity of BRCA-1. The present
invention
satisfies this need and provides related advantages as well.
SUMMARY OF THE INVENTION
The invention is directed to methods of identifying genes that contribute to
the formation of cancer and to the identified genes and gene products as
therapeutic
targets for the treatment of cancer. The methods are directed to the
identification of
genes encoding proteins that regulate the tumor suppressor, BRCA-1, resulting
in an
increased propensity for cancer. Accordingly, the present invention provides
ribozymes and encoding nucleic acids having target recognition sequences that
enable the ribozymes to bind and cleave BRCA-1 regulators, resulting in
upregulation or downregulation of BRCA-1 in a cell.
Also provided are nucleic acids encoding BRCA-1 regulators that contain the
target sequences recognized by the ribozymes of the invention. Fragments of
these
nucleic acid and protein sequences also are provided.
Further provided is a method for identifying a gene for which the expression
level is affected by a BRCA-1 regulator comprising: a) hybridizing a first
mRNA
and a second mRNA to at least one candidate gene, wherein the first mRNA is
obtained from cells expressing a ribozyme that targets and cleaves mRNA
encoding
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a BRCA-1 regulator and wherein the second mRNA is from control cells otherwise
similar to those expressing the ribozyme except that the BRCA-1 regulator mRNA
is
not targeted by a ribozyme; and b) comparing the relative amounts of the first
and
second mRNA that hybridizes to the gene. The mRNAs may be reverse transcribed
into DNA before hybridization and the gene or the mRNAs (or cDNA copies) may
be labeled with a detectable moiety such as a fluorescent dye. In a further
embodiment, hybridization to multiple genes is achieved by arraying the genes
on a
solid support.
Yet further provided is a method of identifying a compound that modulates
the activity of a BRCA-1 regulator, the method comprising contacting a BRCA-1
regulator with a test compound and a target molecule responsive to the
activity of the
BRCA-1 regulator, wherein an increase or decrease in the activity of the BRCA-
1
regulator for the target molecule in the presence of the test compound as
compared to
the absence of the test compound identifies a compound that modulates the
activity
of a BRCA-1 regulator. The activity of the BRCA-1 regulator can be a DNA
binding
activity, protein kinase activity, GTP binding activity, protease activity, or
protein
binding activity.
Still further provided is a method of treating cancer, comprising contacting a
cancer cell with an expression vector that encodes a ribozyme selectively
reactive
with an RNA encoding a BRCA-1 regulator or contacting the cancer cell with the
ribozyme.
Also provided is a method of detecting a neoplastic cell in a sample wherein
the neoplastic cell is associated with an altered expression of a BRCA-1
regulator or
an altered structure of a BRCA-1 regulator as compared to a normal cell,
comprising:
(a) contacting the sample with a detectable agent specific for a BRCA-1
regulator
nucleic acid or an encoded BRCA-1 regulator polypeptide; and (b) detecting the
nucleic acid or polypeptide in the sample, wherein altered expression or
structure of
the polypeptide indicates the presence of a neoplastic cell in the sample.
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BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a haripin ribozyme and a substrate RNA. (SEQ ID NOS: 3
and 99).
Figure 2 shows alignments of the (a) nucleic acid; and (b) amino acid
sequences for BR1 (SEQ ID NOS: 1 and 2, respectively) and Thema EFG (SEQ ID
NOS: 96 and 97, respectively).
Figure 3 shows rounds of cell sorting in selection for cells with increased
EGFP expression.
Figure 4 shows the enrichment of EGFP expressing cells after rounds of cell
sorting.
Figure 5 shows a RNA expression levels after a fifth round of cell sorting.
Figure 6 shows a schematic of a method for amplifying DNA corresponding
to the sequence of a TST.
Figure 7 shows an RST for BR1 and sites of binding for several RSTs of BR1
(SEQ 117 NOS: 100 and 101, respectively).
Figure 8 shows validation ribozyme sequences (SEQ ID NOS: 102 through
221 ).
Figure 9 show the nucleic acid sequence of BR1 (SEQ ID NO: 1) and
predicted translated polypeptide sequence (SEQ ID NOS: 2 and 222 through 231).
DETAILED DESCRIPTION OF THE INVENTION
The invention is directed to methods of identifying genes that contribute to
the formation of cancer and to the identified genes and gene products as
therapeutic
targets for the treatment of cancer. The methods are directed to the
identification of
genes encoding proteins that regulate the tumor suppressor, BRCA-1, resulting
in an
increased propensity for cancer. Genes encoding BRCA-1 regulators are sought
for
identification and as therapeutic targets because decrease in the level of
BRCA-I
expression or BRCA-1 activity is linked with certain cancers, for example,
breast
carcinomas.
In one embodiment, BRCA-1 regulators have been identified. BRCA-1 is a
tumor suppressor which prevents cells from undergoing uncontrolled growth such
as
occurs with cancer cells. BRCA-1 can act as a transcription factor, and thus
BRCA-1
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tumor suppressor activity can result, at least in part, from regulation of
gene
expression. Decreased BRCA-1 activity is associated with cancers such as
breast
cancer, ovarian cancer, endometrial cancer, prostate cancer, and the like.
While in
some cancers, the decreased BRCA-1 activity is due to mutations in the BRCA-1
gene, in other cancers, decreased BRCA-1 activity is due to reduced levels of
BRCA-
1 expression. To date, regulation of BRCA-1 expression has not been well
characterized. In the present invention, numerous nucleic acid sequences have
been
identified which correspond to BRCA-1 regulators. These nucleic acid sequences
have been shown to regulate BRCA-1 gene expression or activity. Ribozymes that
inhibit expression of many of these nucleic acid sequences, including BBC1,
BR1,
>D4, BR2, and BR3, can increase BRCA-1 expression or activity.
As used herein, the term "substantially pure"' when used in reference to .a
nucleic acid or polypeptide of the invention is intended to mean a molecule
that is in
a form that is relatively free from cellular components such as lipids,
polypeptides,
nucleic acids or other cellular material that it is associated with in its
natural state.
As used herein, the term "nucleic acid" is intended to mean a single- or
double-stranded DNA or RNA molecule. For example, a nucleotide designated as
"T" also is equivalent to a "U" nucleotide in a recited sequence. A nucleic
acid
molecule of the invention can be of linear, circular or branched
configuration, and
can represent either the sense or antisense strand, or both, of a native
nucleic acid
molecule. Unless otherwise indicated, a reference to a nucleotide sequence of
a
nucleic acid molecule includes the sequence in single stranded form and in
double
stranded form. The term also is intended to include nucleic acid molecules of
both
synthetic and natural origin. A nucleic acid molecule of natural origin can be
derived from any animal, such as a human, non-human primate, mouse, rat,
rabbit,
bovine, porcine, ovine, canine, feline, or amphibian, or from a lower
eukaryote, such
as Drosophila, C. elegans or yeast. A synthetic nucleic acid includes, for
example,
nucleic acids prepared by chemical and enzymatic synthesis. The term "'nucleic
acid" is similarly intended to include analogues of natural nucleotides which
have
similar binding properties as the referenced nucleic acid and which can be
utilized in
a manner similar to naturally occurring nucleotides and nucleosides.
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As used herein, the term "fragment" when used in reference to a nucleic acid
is intended to mean a portion or segment of the nucleic acid molecule having
the
ability to selectively hybridize or bind to the subject nucleic acid, or its
complement.
The term "selectively hybridize" as used herein, refers to the ability of a
nucleic acid
or fragment to bind the subject nucleic acid molecule without substantial
cross-
reactivity with a molecule that is not the subject nucleic acid molecule.
A fragment of a nucleic acid molecule of the invention includes at least about
8-12 nucleotides of the subject nucleic acid. For example, a ribozyme sequence
tag
(RST) of a hairpin ribozyme and its complementary target sequence tag (TST)
described herein generally include about 14-16 nucleotides of which 6-8
nucleotides
are in helix 1, about 4 nucleotides are in helix 2, the two helixes separated
by 4 non-
base pairing nucleotides. Not all of the RST nucleotides in helix 1 need be
complementary to the TST for the ribozyme to target and cleave the TST
containing
RNA. Generally, 1-2 bases of RST helix 1, depending on the position of the
nucleotide in the sequence, may be non-complementary to the TST without loss
of
ribozyme function, although in some cases, up to 4 of bases may be non-
complementary. Therefore, a fragment having the ability to selectively
hybridize can
contain about 8, 9, 10, 11 or 12 nucleotides of the subject nucleic acid. A
fragment
can also contain a greater number of nucleotides corresponding to the subject
nucleic
acid, or complement thereof, including for example, about 13, 14 or 15
nucleotides
as well as at least 16, 17, 18, 19 or 20 nucleotides so long as it maintains
the ability
to selectively hybridize to the subject nucleic acid. Additionally, a fragment
can be
longer, including at least about 25, 30, 40, 50, 100, 300 or 500 or more
nucleotides,
and can include up to the full length of the reference nucleic acid molecule
minus
one nucleotide. Fragments of such lengths are able to selectively hybridize
with the
subject nucleic acid molecule in a variety of detection formats described
herein and
known to those skilled in the art.
Therefore, a fragment of a nucleic acid molecule of the invention can be
used, for example, as an RST to target a ribozyme to a nucleic acid of the
invention
or as a selective inhibitor of uncontrolled growth in a cell having abnormal
BRCA-1
activity or expression levels. A TST can be used as a PCR primer to
selectively
amplify a nucleic acid molecule of-the invention; as a selective primer for 5'
or 3'
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RACE to determine or identify 5' or 3' sequence of a nucleic acid molecule
identified
in methods of the invention; as a selective probe to identify or isolate a
nucleic acid
molecule of the invention on a RNA or DNA blot, or from a genomic or cDNA
library.
The term "unique" when used in reference to a specific nucleic acid fragment
is intended to mean a fragment of the subject nucleic acid that contains at
least one
nucleotide at a particular position that is characteristic, distinct or novel
when
compared to a different nucleotide sequence, or a related nucleotide sequence
at the
same or analogous position. In reference to a particular sequence, for
example, the
nucleotide sequence encoding the BRCA-1 regulator 1, or BRl (SEQ ID NO: 1)
differs from the elongation factor G (EFG) encoding sequence at about 285
codon
positions or about 694 nucleotides within the coding region. Therefore, for
each of
these codon positions, there is at least one nucleotide which differs from the
EFG
sequence at that position and is therefore characteristic of the BR1
nucleotide
sequence. A BR1 nucleic acid fragment containing one such characteristic
nucleotide is a unique fragment.
As used herein, the term "substantially the same" when used in reference to a
nucleotide sequence is intended to mean a nucleic acid molecule that retains
its
ability to selectively hybridize to the reference nucleic acid. Therefore, a
nucleic
acid molecule having substantially the same sequence compared to a reference
nucleic acid can include, for example, one or more additions, deletions or
substitutions with respect to the reference sequence so long as it can
selectively bind
to that sequence. Included within this definition are encoding nucleic acids
that have
degenerate codon sequences at one or more positions and therefore differ in
nucleotide sequence compared to the reference nucleic acid but substantially
maintain the referenced encoded amino acid sequence.
As used herein the term "substantially the same," when used in reference to a
polypeptide of the invention, is intended to mean an amino acid sequence that
contains minor modifications with respect to the reference amino acid
sequence, so
long as the polypeptide retains one or more of the functional activities
exhibited by
the polypeptide as a whole. A polypeptide that has substantially the same
amino acid
sequence as a reference human amino acid sequence can be, for example, a
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homologous polypeptide from a vertebrate species, such as a non-human primate,
mouse, rat, rabbit, bovine, porcine, ovine, canine, feline, or amphibian, or
from a
lower eukaryote, such as Drosophila, C. elegans or yeast.
A polypeptide that has substantially the same amino acid sequence as a
reference sequence can also have one or more deliberately introduced
modifications,
such as additions, deletions or substitutions of natural or non-natural amino
acids,
with respect to the reference sequence. Those skilled in the art can determine
appropriate modifications that, for example, serve to increase the stability,
bioavailability, bioactivity or immungenicity of the polypeptide, or
facilitate its
purification, without altering the desired functional activity. For example,
introduction of a D-amino acid or an amino acid analog, or deletion of a
lysine
residue, can stabilize a polypeptide and reduce degradation. Likewise,
addition of
tag sequences, such as epitope or histidine tags, or sorting sequences, can
facilitate
purification of the recombinant polypeptide. Depending on the modification and
the
source of the polypeptide, the modification can be introduced into the
polypeptide, or
into the encoding nucleic acid sequence.
Computer programs known in the art, for example, DNASTAR software,
can-be used to determine which amino acid residues can be modified as
indicated
above without abolishing the desired functional activity. Additionally,
guidance in
modifying amino acid sequences while retaining functional activity is provided
by
aligning homologous BRCA-1 regulator polypeptides from various species. Those
skilled in the art understand that evolutionarily conserved amino acid
residues and
domains are more likely to play a role in the biological activity than less
well-
conserved residues and domains.
In general, an amino acid sequence that is substantially the same as a
reference amino acid sequence will have greater than about 25% identity with
the
reference sequence, although an amino acid sequence that is substantially the
same
as a reference sequence can have greater than about 30% identity, greater than
about
40% identity, greater than about 50% identity, greater than about 60%
identity,
greater than about 70% identity, preferably greater than about 80% identity,
more
preferably greater than about 90% identity, and especially preferably greater
than
about 95% identity, or greater than about 98% identity. The amino acid
sequences
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which align across two sequences, and the presence of gaps and non-homologous
regions in the alignment, can be determined by those skilled in the art based,
for
example, on a BLAST 2 or Clustal V or similar computer alignment software. A
computer alignment can, if desired, be optimized visually by those skilled in
the art.
The percent identity of two sequences is determined as the percentage of the
total
amino acids that align in such an alignment which are identical. Those skilled
in the
art understand that two amino acid molecules with a given percentage identity
over
the entire sequence or over a substantial portion or portions thereof, are
more likely
to exhibit similar functional activities than two molecules with the same
percentage
identity over a shorter portion of the sequence. Sequence identity is
preferably
determined with by BLAST searching with the default settings provided at the
website of the National Cancer Biological Institute (NCBI).
As used herein, the term "functional fragment" when used in reference to a
polypeptide of the invention is intended to refer to a portion, segment or
fragment of
the polypeptide, which retains at least one of the activities of the full
length
polypeptide. For example, a functional fragment of any of the BRCA-1
regulators of
the invention can be a portion of the polypeptide that maintains its ability
to regulate
BRCA-1 expression or activity. For the specific example of >D4, a functional
fragment of >D4 can be a portion of >D4 that maintains its ability to bind
with one or
more helix-loop-helix transcription factors or a portion of 1D4 that modulates
differentiation in adipocytes or neuronal cells.
As used herein, the term "ribozyme sequence tag" or "RST" is intended to
mean the target recognition domain of a ribozyme. Therefore, the structure of
an
RST hairpin ribozyme can be 5'-N8-AGAA-N4-3' where Ng and N4 are
complementary to sequences of the target RNA . The bases AGAA form a non-
binding loop with the NGUC sequence of the target RNA. Therefore, a "target
sequence tag" nucleic acid or "TST" as used herein, is a nucleic acid having a
nucleotide sequence that is capable of selectively hybridizing to an RST of a
ribozyme and being cleaved by the ribozyme. For example, the TST regions
capable
of selectively hybridizing to the RST will be substantially the complement of
the
helix 1 and helix 2 RST region sequences. These selectively hybridizing
regions are
separated by, for example, a GUC which is capable of being cleaved by a
hairpin
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ribozyme and therefore will have the structure 5'-NS-GUC-N8-3' where the first
four
nucleotides of NS represent the TST complementary sequence of the ribozyme
helix
2 and Ng represents the TST complementary sequence of the ribozyme helix 1.
As used herein, the term "ribozyme" or "ribozyme RNA molecule" is
5 intended to mean a catalytic RNA that cleaves RNA. Ribozymes include both
hairpin and hammerhead classes, which differ in mechanism for hybridization.
The
term "hairpin ribozyme" is intended to refer to an RNA molecule having the
general
nucleic acid sequence and two-dimensional configuration of the molecule shown
in
Figure 1, which is capable of selectively hybridizing, or of both selectively
10 hybridizing and cleaving, a target RNA. The term is also intended to
include both
hairpin ribozyme RNA molecules as well as single- and double-stranded DNA
molecules that, when expressed, form hairpin ribozyme RNA molecules.
Generally,
a hairpin ribozyme will have from about 50 to 54 nucleotides, which form two
helical domains (helix 3 and helix 4) and 3 loops (Loops 2, 3 and 4). Two
additional
helices, helix 1 and helix 2, form between the ribozyme and its RNA target or
substrate (Figure 1). A hairpin ribozyme binds a target RNA by forming Watson-
Crick base pairs between the substrate and helix 1 and helix 2 sequences (see
dots in
Figure 1; "N" is any nucleotide). The length of helix 2 is usually about 4
nucleotides, and the length of helix 1 can vary from about 6-10 nucleotides or
more.
A hairpin ribozyme can have catalytic activity, and thus cleave the target RNA
such
as at the cleavage site shown in Figure 1. The catalytic activity of the
hairpin
ribozyme also can be disabled by, for example, altering the AAA sequence in
Loop 2
to CGU. Those skilled in the art can determine which modifications to the
overall
hairpin ribozyme structure can be made and still maintain the target binding,
or both
target binding and catalytic activity of a hairpin ribozyme of the invention.
As used herein, the term "library" or "ribozyme library" is intended to mean a
collection or population of different species of ribozyme RNA molecules.
Within a
population, any of the ribozyme species can be uniquely represented or
redundant.
Therefore, the term "randomized" or "random" when used in reference to a
ribozyme
library is intended to refer to a population of ribozymes that have differing
nucleotide sequences in their target recognition sequence. The differing
nucleotide
sequences can be purposefully introduced, such as by degenerate, partially
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degenerate or variegated oligonucleotide synthesis, or other methods well
known to
those skilled in the art. Alternatively, the differing nucleotide sequences
can be
introduced by a variety of mutagenesis methods including, for example,
chemical
and enzymatic methods well known in the art. A random ribozyme library also
can
be assembled, for example, from combining a collection of different ribozyme
species into a single population. Details on the synthesis and construction of
random
ribozyme libraries is described in the Examples and in WO 00/05415 to Barber
et al.
As used herein, the term "target recognition sequence" when used in
reference to a ribozyme is intended to mean the substrate binding site of a
ribozyme,
which corresponds to an RST RNA nucleotide sequence or which corresponds to
the
complement of an TST nucleic acid nucleotide sequence. For the specific
example
of a hairpin ribozyme, the target recognition sequence corresponds to the
nucleotide
sequences of the helix 1 or helix 2 domain or both (see Figure 1). The target
recognition sequences of helix 1 and 2 can be separated by catalytic
nucleotides,
which in the specific example of a hairpin ribozyme correspond to the
nucleotides
AGAA.
As used herein, the term "BRCA-1 regulator" is intended to mean a gene
product or a nucleic acid, such as a structural or functional RNA, that
modulates the
expression or activity of BRCA-1. A BRCA-1 regulator can modulate BRCA-1
expression through, for example, direct binding of a BRCA-1 regulator to a
nucleic
acid element that influences BRCA-1 expression, such as the BRCA-1 5'
regulatory
region, where the BRCA-1 regulator bound to the nucleic acid element results
in an
increase or decrease in BRCA-1 expression. Another manner iri which a BRCA-1
regulator can modulate BRCA-1 expression is by binding to a separate molecule
that
modulates BRCA-1 expression, such as a transcription factor, in such a way
that
results in an increase or decrease in BRCA-1 expression. BRCA-1 expression can
also be modulated by a BRCA-1 regulator which increases or decreases the
translation of BRCA-1-encoding mRNA. Additionally, a BRCA-1 regulator can
bind to BRCA-1, thereby modulating BRCA-1 activity. For example, a BRCA-1
regulator can bind BRCA-1 thereby increasing or decreasing the transcriptional
activation activity of BRCA-1. Similarly, BRCA-1 regulator can modulate BRCA-1
activity by binding a separate molecule that modulates BRCA-1 expression, for
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example a transcription factor such as TFIIH, and thereby increase or decrease
BRCA-1 activity. It is not necessary that the BRCA-1 regulator be essential
for
modulation of BRCA-1 expression or activity. All that is needed is that the
BRCA-1
regulator be functionally involved in modulating BRCA-1 expression or
activity. A
BRCA-1 regulator can be encoded by a gene including, for example, a gene
originating from the species of the infected cell type as well as a
heterologous gene
that becomes incorporated into the cell's genome. Also, in accordance with the
invention, regulators of BRCA-1 can be involved in regulating BRCA-2 or other
factors involved in the DNA repair response.
As used herein, the term "modulate" when used in reference to BRCA-1
expression or activity refers to the ability of a BRCA-1 regulator to alter
BRCA-1
expression or activity. Altered expression or activity includes, for example,
an
increase or decrease in BRCA-1 expression or activity.
As used herein, the term "BRCA-1" is intended to mean a member of the
BRCA-1 tumor suppressor family. Exemplary members of the tumor suppressor
family are human BRCA-1 and species homologs of BRCA-1.
As used herein, the term "selection marker" means a natural or modified gene
product that influences cell viability or cell growth, or can be used as a
measurable
indicator of an activity associated with a cell such as viability, growth,
gene
expression and the like. Specific examples include enhanced green fluorescence
protein (EGFP), hygromycin resistance gene and the tumor suppressor BRCA-1.
For
example, in the presence of hygromycin, the expression of the hygromycin
resistance
gene permits cells to survive. In a contrasting example, expression of BRCA-1
suppresses anchorage-independent cell growth, resulting in, for example,
decreased
ability to grow in soft agar. EGFP is another selection marker that can be
used as a
measurable indicator of gene expression. Use of EGFP allows a specific, user
defined expression level to be selected for fluorescence activated cell
sorting (FACS)
and other cell analysis methods. Iterative FACS-based selection results in
enrichment of cells expressing EGFP at the user-defined expression level.
As used herein, the term "treating" when used in reference to cancer is
intended to mean a reduction in the severity, or prevention of a neoplastic
disease.
Therefore, "'treating a cancerous disease" as used herein, is intended to mean
a
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reduction in severity, regression, or prevention of a cancerous disease.
Reduction in
severity includes, for example, an arrest or a decrease in clinical symptoms,
physiological indicators or biochemical markers. Prevention of the cancer
includes,
for example, precluding the occurrence of the cancer, such as in prophylactic
uses to
individuals having a known or suspected propensity for developing cancer, or
reversing the cancer in an individual to their non-diseased state of health.
As used herein, the terms "cancer," "tumor" and "neoplasm" and grammatical
variants thereof are intended to mean abnormal growth in a tissue or organ.
Typically, such a growth is characterized by uncontrolled cell proliferation
and can
be malignant or benign.
As used herein, the term "neoplastic cell" is intended to mean a cell that has
altered expression or activity of a BRCA-1 regulator compared to a normal cell
from
the same or a different individual. A neoplastic cell will generally also
exhibit
histological or proliferative features of a malignant or premalignant cell.
For
example, histological methods can be used to show invasion of surrounding
normal
tissue, increased mitotic index, increased nuclear to cytoplasmic ratio,
altered
deposition of extracellular matrix, or a less differentiated cell phenotype. A
neoplastic cell can also exhibit unregulated proliferation, such as anchorage
independent cell growth, proliferation in reduced-serum medium, loss of
contact
inhibition, or rapid proliferation compared to normal cells.
As used herein, the term "altered expression" of a BRCA-1 regulator nucleic
acid detected by a method of the invention refers to an increased or decreased
amount of a BRCA-1 regulator nucleic acid in the test sample relative to known
levels in a normal sample. Altered abundance of a nucleic acid molecule can
result,
for example, from an altered rate of transcription, from altered transcript
stability, or
from altered copy number of the corresponding gene.
As used herein, the term "altered structure" of a nucleic acid molecule refers
to differences, such as point mutations, deletions, translocations, splice
variations
and other rearrangements, between the structure of a BRCA-1 regulator nucleic
acid
molecule in a test sample and the structure of the BRCA-1 regulator nucleic
acid
molecule in a normal sample. Those skilled in the art understand that
mutations that
alter the structure of a nucleic acid molecule can also alter its expression.
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As used herein, the term "altered expression" of a BRCA-1 regulator
polypeptide refers to an increased or decreased amount, or altered subcellular
localization of the polypeptide in the test sample relative to known levels or
localization in a normal sample. Altered abundance of a polypeptide can
result, for
example, from an altered rate of translation or altered copy number of the
corresponding message, or from altered stability of the protein. Altered
subcellular
localization can result, for example, from truncation or inactivation of a
sorting
sequence, from fusion with another polypeptide sequence, or from altered
interaction
with other cellular polypeptides.
As used herein, the term "altered structure" of a polypeptide refers to
differences in amino acid sequence, post-translational modifications, or
conformation, of the polypeptide in the test sample relative to a normal
sample which
result in altered expression or activity of a BRCA-1 regulator polypeptide.
Post-
translational modifications include, for example, phosphorylation,
glycosylation and
acylation. Such differences can be detected, for example, with a structure-
specific
detectable binding agent.
As used herein, the term "sample" is intended to mean any biological fluid,
cell, tissue, organ or portion thereof, that includes or potentially includes
nucleic
acids and polypeptides of the invention. The term includes samples present in
an
individual as well as samples obtained or derived from the individual. For
example,
a sample can be a histologic section of a specimen obtained by biopsy, or
cells that
are placed in or adapted to tissue culture. A sample further can be a
subcellular
fraction or extract, or a crude or substantially pure nucleic acid or protein
preparation. A sample can be prepared by methods known in the art suitable for
the
particular format of the detection method.
As used herein, the term "detectable agent" refers to a molecule that renders
a
BRCA-1 regulator nucleic acid or polypeptide detectable by an analytical
method.
An appropriate detectable agent depends on the particular detection format,
and can
be determined for a particular application of the method by those skilled in
the art.
For example, a detectable agent specific for a BRCA-1 regulator nucleic acid
molecule can be a complementary nucleic acid molecule, such as a hybridization
probe or non-catalytic ribozyme, that selectively hybridizes to the nucleic
acid
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molecule. A hybridization probe or ribozyme can be labeled with a detectable
moiety, such as a radioisotope, fluorochrome, chemiluminescent marker, biotin,
or
other detectable moiety known in the art that is detectable by analytical
methods.
A detectable agent specific for a BRCA-1 regulator nucleic acid molecule can
5 also be, for example, a PCR or RT-PCR primer, which can be used to
selectively
amplify all or a desired molecule, which can then be detected by methods known
in
the art. Furthermore, a detectable agent specific for a BRCA-1 regulator
nucleic acid
molecule can be a selective binding agent, such as a peptide, nucleic acid
analog, or
small organic molecule, identified, for example, by affinity screening of a
library of
10 compounds.
A detectable agent specific for a BRCA-1 regulator polypeptide can be, for
example, an agent that selectively binds the polypeptide. For example, such a
detectable agent is one that selectively binds with high affinity or avidity
to the
polypeptide without substantial cross-reactivity with other polypeptides that
are not
15 BRCA-1 regulator polypeptides. The binding affinity of a detectable agent
that
selectively binds a polypeptide will generally be greater than about 10-5 M
and more
preferably greater than about 10-6 M for the polypeptide. High affinity
interactions
are preferred, and will generally be greater than about 10-g M to 10-9 M.
A detectable agent specific for a BRCA-1 regulator polypeptide can be, for
example, a polyclonal or monoclonal antibody specific for the polypeptide, or
other
selective binding agent identified, for example, by screening a library of
compounds
for binding to the regulator polypeptide. For certain applications, a
detectable agent
can be utilized that preferentially recognizes a particular conformational or
post-
translationally modified state of the polypeptide. The detectable agent can be
labeled
with a detectable moiety, if desired, or rendered detectable by specific
binding to a
detectable secondary binding agent.
The invention provides a substantially pure nucleic acid comprising a
nucleotide sequence greater than about 57% identical to SEQ >D NO: 1, or a
unique
fragment thereof. Also provided is a substantially pure polypeptide comprising
an
amino acid sequence greater than about 41% identical to SEQ >D NO: 2, or
functional fragment thereof.
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16
The nucleic acid shown as SEQ ID NO: 1 has been found to encode a BRCA-
1 regulator, which functions in reducing the expression of BRCA-1. As such, it
is
useful as a target for treating or reducing the severity of a cancer by
regulating its
proliferation through modulating BRCA-1 expression. Inhibition of the
expression
or activity of a BRCA-1 regulator results in a concomitant increase in BRCA-1
expression and therefore will result in decreased tumor cell proliferation.
SEQ ID NO: 1 corresponds to the expressed message of the human gene
encoding BRCA-1 regulator termed BRCA-1 regulator 1 (BR1). SEQ ID NO: 1 is
about 2967 nucleotides in length and has 5' and 3' non-coding regions of 395
and 988
nucleotides, respectively (Figure 9). The resultant coding region is 1584
nucleotides
in length, coding for a polypeptide of 521 amino acids. SEQ ID NO: 1 has a
nucleotide sequence of about 57% identical to the human EFG sequence.
Modifications of SEQ ID NO: 1, which do not substantially affect the activity
of the encoded BRCA-1 regulator and which maintain nucleotide sequence
identity
greater than about 57% are included as nucleic acids of the invention. These
nucleic
acids having minor modifications can similarly be used for the development of
therapeutic compounds which inhibit the expression or activity of BR1. Such
modifications include, for example, changes in the nucleotide sequence which
do not
alter the encoded amino acid sequence as well as changes in the nucleotide
sequence
resulting in conservative amino acid substitutions or minor alterations which
do not
substantially affect the BRCA-1 regulating activity of BR1. Those skilled in
the art
will known or can determine what changes within greater than about 57%
compared
to SEQ ID NO: 1 can be made without substantially affecting the activity of
BR1 as
a regulator of BRCA-1 expression or activity.
Unique fragments of SEQ ID NO: 1 are also provided. The fragments are
useful in a variety of procedures, including for example, as probes for
determining
the effectiveness of therapeutic agents which target expression of BR1 as a
regulator
of BRCA-1 expression or activity. Unique fragments also can be used to encode
functional fragments of BR1 as therapeutic targets for anti-cancer compounds
in the
screening methods of the invention. The unique fragments of SEQ ID NO:1 are
applicable in a variety of other methods and procedures known to those skilled
in the
art.
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17
Unique fragments of SEQ ID NO: 1 correspond to fragments or portions of
SEQ m NO: 1 that are of sufficient length to distinguish the fragment as a BR1
encoding nucleic acid and that contain at least one nucleotide characteristic
of SEQ
ID NO: 1. Such a characteristic nucleotide, or nucleotides, within a specific
fragment of SEQ ID NO: 1 distinguish that fragment from other related
nucleotide
sequences. For example, fragments of the non-coding region of SEQ >D NO: 1 are
generally unique when compared to nucleotide sequences such as EFG, for
example,
because there is little evolutionary pressure to conserve non-coding domains.
Nucleic acid sequences as small as between about 12-15 nucleotides are
statistically
unique sequences within the human genome. However, nucleic acids as small as
between about 8-12 nucleotides can be unique. Therefore, non-coding region
fragments of SEQ ID NO: 1 of about 8-9, preferably about 10-11, and more
preferably about 12 or 15 nucleotides or more in length can be nucleotide
sequences
corresponding to unique fragments of SEQ ID NO: 1 of the invention.
Additionally, unique nucleotide sequences arise in the coding region of SEQ
ID NO: 1 as well. Those skilled in the art will know or can determine which
nucleotide positions are unique to either a non-coding region or a coding
region
fragment of SEQ >D NO: 1 given the teachings described herein or by alignment
of
SEQ ID NO: 1 with other sequences to be distinguished using methods well known
to those skilled in the art. The nucleic acid sequence alignment of SEQ >D NO:
1
with the nucleic acid sequence of EFG shows that there are about 285 codon
differences between the BR1 and EFG sequences indicating ~at least about 694
or
more unique nucleotides in SEQ ~ NO: 1 compared to the EFG encoding nucleotide
sequence (Figure 2). Inclusion of any or all of these distinguishing
nucleotide
differences within a fragment of SEQ ID NO: 1 confers uniqueness onto the
fragment.
A substantially pure nucleic acid molecule having a nucleotide sequence
greater than about 57% identical to SEQ ID NO: 1, or a unique fragment
thereof, will
be of sufficient length and identity to SEQ 117 NO: 1 to selectively hybridize
to it
under at least moderately stringent hybridization conditions. For example, it
can be
determined that a substantially pure nucleic acid molecule contains a
nucleotide
sequence greater than about 57% as SEQ ID NO: 1, or a unique fragment thereof,
by
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18
determining its ability to hybridize in a filter hybridization assay to a
molecule
having the sequence of SEQ ID NO: 1, but not to other unrelated nucleic acid
molecules, under conditions equivalent to hybridization in 50% formamide, 5X
Denhart's solution, 5X SSPE, 0.2% SDS at 42°C, followed by washing
in 0.2X
SSPE, 0.2% SDS, at 65°C. Suitable alternative buffers and hybridization
conditions
that provide for moderately stringent hybridization conditions in particular
assay
formats are known or can be determined by those skilled in the art (see, for
example,
Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor
Laboratory Press, 1989).
The nucleic acid shown as SEQ ID NO: 1 encodes a polypeptide having the
amino acid sequence shown in SEQ ID NO: 2. As with the nucleotide sequence
described above, modifications of SEQ ID NO: 2 which do not substantially
affect
the activity of the BRCA-1 regulator and which maintain amino acid sequence
identity greater than about 41% are included as polypeptides of the invention.
These
polypeptides having minor modifications can similarly be used for the
development
of therapeutic compounds which inhibit the BRCA-1-regulating activity of BRl.
Such modifications include, for example, changes in the amino acid sequence
which
do not substantially alter the structure or function of a domain within the
polypeptide
as well as changes in the amino acid sequence which result in conservative
substitutions or minor alterations which do not substantially affect the
activity of
BR1. Those skilled in the art will known or can determine what changes within
greater than about 41% compared to SEQ ID NO: 2 can be made without
substantially affecting the BRCA-1-regulating activity of BR1.
Functional fragments of SEQ ID NO: 2 are also provided. The BRCA-1
regulator BR1 can regulate BRCA-1 by inhibiting the translation of BRCA-1
mRNA.
Therefore, it is within the scope of the invention that an example of a
functional
fragment of BR1 is a domain that inhibits translation of a mRNA into a
polypeptide,
for example the, translation of BRCA-1 mRNA into a polypeptide.
The invention also provides a substantially pure TST nucleic acid including a
fragment having substantially the nucleotide sequence of SEQ ID NO: 1, wherein
the
TST has substantially the nucleotide sequence 5'-N5-GUC-N$-3' or 5'-NS-GUA-
Ng-3' (SEQ ID NOS: 3 and 4, respectively). The TST nucleic acid portion of the
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19
fragment can have between about 8-12 nucleotides, and preferably about 9-10
nucleotides at positions NS and N8, that are identical to a fragment of SEQ )D
NO: 1.
Therefore, depending on the length of the TST nucleic acid portion, a fragment
of
SEQ ID NO: 1 as described above can be about 11-15 nucleotides or greater in
length.
Hairpin ribozymes cleave RNA substrates 5' to the G nucleotide in either of
the sequences 5'-NS-GUC-Ng-3' or 5'-NS-GUA-N$-3' (SEQ )D NOS: 3 and 4,
respectively) Fragments of SEQ ID NO: 1 having a corresponding RNA form that
is
recognized by the target recognition site of a hairpin ribozyme therefore
include
nucleic acids having the sequence 5'-NS-GUC-Ng-3' or 5'-NS-GUA-Ng-3' (SEQ ID
NOS: 3 and 4, respectively) where NS and N8 are nucleotide sequences
substantially
the same as a sequence corresponding to SEQ ID NO: 1. Such fragments
correspond
to the complement sequence of a ribozyme target recognition site or RST, and
are
referred to herein as TST nucleic acids.
The TST nucleic acids can be of any desired length and can include
additional sequences other than those corresponding to SEQ ID NO: 1 and other
moieties so long as they have the structure 5'-NS-GUC-N$-3' or 5'-NS-GUA-N8-3'
(SEQ ID NOS: 3 and 4, respectively) where NS and Ng correspond substantially
to a
nucleotide sequence of SEQ >D NO: 1. Moreover, it is not necessary for all
nucleotide residues within the NS and Ng regions to be identical to the
corresponding
sequence within SEQ ID NO: 1. Instead, all that is necessary is for such TST
nucleic
acids to selectively hybridize to a complementary RST. Therefore, less than
all 13
nucleotides at positions NS and Ng can be identical to a nucleotide sequence
or
fragment of SEQ ID NO: 1. Generally, between about 8-12 or between about 9-10
nucleotides are sufficient for selective hybridization of a RST with a TST
nucleic
acid. Described further below in the Examples is a specific example of an RST
present in a ribozyme that selectively hybridizes to SEQ ID NO: 1.
Similarly, the TST nucleic acids can be used to design ribozymes that
selectively hybridize and cleave a RNA corresponding to SEQ ID NO: 1. A
specific
example of such a ribozyme is a hairpin ribozyme having a target recognition
sequence complementary to a TST nucleic acid of SEQ ID NO: 1 and having the
nucleotide sequence 5'-Ng-AGAA-N4-3'. As with the TST nucleic acids described
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above, it is not necessary that all the sequences with position N8 and N4 be
identical
in complement to SEQ >D NO: 1 so long as the target recognition sequence can
selectively hybridize to the RNA form of TST nucleic acid and cleave it as a
substrate. Those skilled in the art will know or can determine given the
teachings
5 and descriptions herein what RST sequences are sufficient for selective
hybridization
as well as for cleavage of a target RNA substrate.
Exemplary ribozymes selective for SEQ >D NO: 1 were constructed as
described in the Examples. These ribozymes have the RST sequences: 5'-
UUGAUGUGAGAAGCUU-3', 5'-ACUUUUCUAGAAGGAA-3', 5'-
10 UAUUCCAUAGAAACUG-3', 5'-AGGACUGGAGAAAGCC-3', 5'-
AACAACAUUAGAAUCAA-3' (SEQ 117 NOS: 17-21).
Therefore, the invention provides a ribozyme having a target recognition
sequence capable of selectively hybridizing to a RNA corresponding to SEQ >D
NO:
1 and cleaving said RNA. The target recognition sequence of the ribozyme can
15 consist of a RST complementary to a fragment of SEQ >D NO: 1 and having
substantially the nucleotide sequence 5'-N8-AGAA- N4-3' (SEQ ID NO: 99). The
target recognition sequence can further be between about 8-12 nucleotides,
preferably about 9-10 nucleotides at positions Ng, and N4 that are
complementary to
a fragment of SEQ >D NO: 1.
20 The invention further provides a RST having one of the following nucleotide
sequences:
5'-CCGGAUGCAGAACAAU-3', 5'-AGUACAUUAGAAUACU-3' S'-
CUAGUGAGAGAAGGGA-3', 5'-UGAGAUCCAGAAAAGC-3'
5'-UGUUACUAGAAUGUU-3', and 5'-CCCUAUUUAGAAUUGU-3' (SEQ ID
NOS: 5-10, respectively), or a complementary sequence thereof as described
further
below. The complementary sequence at positions 9-12 (5'-AGAA-3') can be
substituted by the non-complementary ribozyme cleavage sequence 5'-NGUC-3'.
Libraries of ribozymes containing different RST sequences were expressed in
cells, and cells expressing an increased level of BRCA-1 marker were selected.
SEQ
>D NOS: 5-10 represent RST sequences of ribozyme binding sites which, when the
ribozymes were expressed, resulted in an increased level of BRCA-1 marker
expression. BRCA-1 marker expression was determined by increased EGFP
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21
expression, increased hygromycin resistance or decreased ability to grow in
soft
agar. These assays for BRCA-1 marker expression can be affected by both BRCA-1
promoter activity and BRCA-1 activity, since BRCA-1 itself can modulate BRCA-1
promoter activity. Therefore, the ribozymes containing RST sequences selective
for
a BRCA-1 regulator, such as the RSTs of SEQ ID NOS: 5-10, can increase BRCA-1
marker expression by selectively cleaving RNAs that regulate BRCA-1 promoter
activity, BRCA-1 expression, BRCA-1 activity, or any combination thereof.
Each of the RST sequences SEQ ID NOS: 5-10 correspond to a nucleic acid
encoding a BRCA-1 regulator As described previously with respect to TST
nucleic
acid fragments of the BRCA-1 regulator shown as SEQ >D NO: 1, the 5' terminal
Ng
positions and the 3' terminal NS positions are separated by the intervening
trinucleotide sequence 5'-GUC-3' in the BRCA-1 regulator RNA and correspond to
the complement of the RST sequence or the TST sequence. Also as described
previously, at least about 8-12 nucleotides within positions N$ and NS of a
TST are
sufficient for selective binding between a ribozyme and its BRCA-1 regulator
target
RNA. Therefore, a BRCA-1 regulator nucleic acid molecule of the invention
contains least about 8-12 nucleotides corresponding to a RST sequence set
forth as
SEQ >D NOS: 5-10, or its TST sequence complement corresponding to SEQ ID
NOS: 11-16, and including the intervening trinucleotide 5'-GUC-3'.
Therefore, the invention also provides a TST nucleic acid having one of the
following nucleotide sequences:
5'-AUUGNGUCGCAUCCGG-3', 5'-AGUANGUCAAUGUACU-3',
5'-UCCCNGUCCUCACUAG-3', 5'-GCUUNGUCGGAUCUCA-3',
5'-AACANGUCAGUAACA-3', and 5'-ACAANGUCAAAUAGGG-3'
(SEQ >D NOS: 11-16, respectively), or a complementary sequence thereof.
For simplicity of the description, the BRCA-1 regulator nucleic acids of the
invention will be described with reference to its TST nucleic acid sequence
and
specifically with reference to a BRCA-1 nucleic acid containing at least about
8-12
regulator nucleotides corresponding to a TST nucleic acid sequence of SEQ ID
NOS:
11-16. However, it is to be understood that reference to a BRCA-1 regulator
TST
nucleotide sequence also specifically includes reference to the complementary
sequence of the RST nucleic acid molecule. Therefore, it is also to be
understood
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22
that reference to a BRCA-1 regulator containing at least about 8-12
nucleotides
corresponding to a TST sequence of SEQ ID NOS: 11-16 also includes reference
to
a BRCA-1 regulator nucleic acid containing at least about 11-15 nucleotides
corresponding to a TST sequence set forth as SEQ ID NOS: 11-16, which includes
the intervening trinucleotide 5'-GUC-3' between the 5' terminal N$ positions
and the
3' terminal NS positions and as described previously with respect to the BRCA-
1
regulator shown as SEQ ID NO: 1 and its TST fragments. Therefore, the
invention
also provides substantially pure TST nucleic acids having the structure 5'-NS-
GUC-
Ng-3' or 5'-NS-GUA-N$-3' and substantially the nucleotide sequences shown as
SEQ ID NOS: 11-16.
A BRCA-1 regulator nucleic acid molecule containing at least about 8-12
nucleotides corresponding to a TST nucleic acid sequence set forth as SEQ ID
NOS:
11-16, or about 11-15 nucleotides corresponding to a TST nucleic acid sequence
set
forth as SEQ ID NOS: 11-16, and including the intervening trinucleotide 5'-GUC-
3'
or a functional fragment thereof, does not have the exact endpoints of
nucleotide
sequences deposited and available in public databases. Such databases include,
for
example, the nonredundant GenBank database and NCBI dbest EST database.
A BRCA-1 regulator nucleic acid molecule of the invention containing at
least about 8-12 nucleotides corresponding to a TST sequence set forth as SEQ
ID
NOS: 11-16, can be advantageously used, for example, as a therapeutic target
for the
treatment of cancer, as a diagnostic target for determining the presence or
propensity
for cancer, or to identify and isolate additional sequences corresponding to
other
regions of the BRCA-1 regulator nucleic acid molecules of the invention. When
used for the latter purpose, the nucleic acid molecule can contain none, one,
or many
nucleotides at the 5' or 3' end, or both, of the TST sequences recited as SEQ
ID NOS:
11-16. These additional nucleotides can correspond to the native sequence of
the
BRCA-1 regulator nucleic acid molecule, or can be nonnative sequences, or
both.
For example, non-native flanking sequences that correspond to a restriction
endonuclease site or a tag, or which stabilize the nucleic acid containing at
least
about 8-12 nucleotides corresponding to a TST nucleic acid sequence set forth
as
SEQ-ID NOS: 11-16, in a hybridization assay, can be advantageous when the
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23
nucleic acid molecule is used as a probe or primer to identify or isolate
longer
BRCA-1 regulator nucleic acid molecules.
Native BRCA-1 regulator nucleotide sequences flanking the at least about 8-
12 nucleotides corresponding to a TST sequence set forth as SEQ >D NOS: 11-16,
can be determined by methods known in the art, such as RT-PCR, 5' or 3' RACE,
screening of cDNA or genomic libraries, and the like, using an oligonucleotide
having at least about 8-12 nucleotides corresponding to the TST sequence of
SEQ )D
NOS: 11-16 as a primer or probe, and sequencing the resultant product. The
appropriate source of template RNA or DNA for amplification, extension or
hybridization screening can be determined by those skilled in the art.
A specific example of a substantially pure BRCA-1 regulator nucleic acid
molecule containing at least about 8-12 nucleotides of a TST corresponding to
SEQ
ID NO: 13 and flanking coding sequence is the BRCA-1 regulator nucleic acid
molecule having the nucleotide sequence set forth as SEQ ID NO: 1. The
isolation
of SEQ ID NO: 1, based on knowledge of the corresponding RST sequence of SEQ
ID NO: 7, is described further below iri the Examples. Therefore, such
procedures
can be used to identify and substantially purify longer nucleic acid molecules
that
contain at least about 8-12 nucleotides corresponding to a TST of SEQ ID NOS:
11-
16. Such molecules and their functional fragments can be used to produce BRCA-
1
regulator polypeptides and specific antibodies, for example, by methods known
in
the art and described herein, for use in the diagnostic and therapeutic
methods
described herein and known in the art.
In this regard, SEQ ID NOS: 5, 6, 8 and 9, have similarly been used to
identify flanking nucleic acid sequences of the corresponding encoded BRCA-1
regulators. Specifically, SEQ ID NO: 5 has been used to identify a BRCA-1
regulator which was found to correspond to breast basic conserved protein 1
(BBC1,
GenBank accession number X64707). BBCI, identified previously from a human
breast carcinoma cDNA library, is downregulated in hormone-refractory prostate
cancer. BBC1 contains a 25 amino acid region which has strong similarity to
the
plant basic peptide P14.
SEQ ID NO: 5 also has been used to identify a protein which corresponds to
human CHL1 related protein CHLR2, a partial sequence of which is reported in
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24
GenBank as accession number U33834. A portion of CHLR2 has been reported to
act as a helicase. CHLR2 localizes to the nucleus and is apparently expressed
at high
levels only in proliferating human cell lines.
SEQ ID NO: 8 has been used to identify a further BRCA-1 regulator which
was found to correspond to a polypeptide termed inhibitor dominant negative 4
(>D4,
GenBank accession number NM_001546). ID4 is a transcriptional repressor of
basic
helix-loop-helix transcription factors which prevents transcriptional
activation by
dimerization to transcription factors, thereby blocking the ability of the
transcription
factor to bind DNA. It is within the scope of the invention that ID4 can bind
to a
transcription factor that increases BRCA-1 expression, including BRCA-1.
SEQ ID NO: 8 also has been used to identify protein corresponding to ALL-1
fusion partner AF6 (GenBank accession number AB011399), a translation
breakpoint sequence found in acute myeloid leukemia. AF6 also is considered to
be
a Ras-binding protein and regulator of cell junction formation.
SEQ ID NOS: ~6 and 9 have been used to identify BRCA-1 regulators
corresponding to polypeptides termed herein as BR2 and BR3 (GenBank accession
numbers AL045940 and AI273697, respectively), which have been reported only as
EST sequences, and have not been characterized.
SEQ ID NO: 6 also has been used to identify an EST, termed BR4, having
GenBank accession No. AI668913.
As described previously, a BRCA-1 regulator nucleic acid molecule, when
functionally inactivated in a cell, results in increased BRCA-1 expression or
activity,
or both. Such increase results in the concomitant decrease in the ability of a
cell to
proliferate or undergo uncontrolled growth. Similar results can be observed by
inactivation of the BRCA-1 regulator polypeptide by, for example, inhibiting
its
activity. The BRCA-1 regulator activity of a nucleic acid molecule containing
at
least about 8-12 nucleotides corresponding to a TST of SEQ ID NOS: 11-16 and
additional native nucleic acid sequences can be further demonstrated using
various
methods known in the art and described herein. For example, nucleic acid
sequences
flanking the SEQ ID NOS: 11-16 sequences can be selectively targeted in a cell
with
ribozymes by the methods described herein. The effect on cell proliferation
due to
decreased BRCA-1 expression or activity or both can be determined by the
assays
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described below. If inactivation by ribozymal cleavage of a second sequence
within
the isolated nucleic acid molecule also results in increased BRCA-1 expression
or
activity, that nucleic acid molecule is a confirmed BRCA-1 regulator nucleic
acid
molecule.
5 For example, ribozymes targeted toward a second sequence within the
nucleic acid molecules of BBC1, >D4 and BR1, have increased BRCA-1 or reporter
gene expression and/or decreased cell proliferation in soft agar, further
confirming
these genes as encoding BRCA-1 regulators.
Therefore, knowledge of the nucleic acid sequence of a BRCA-1 regulator
10 provides information that can be used to target further ribozymes against
the BRCA-
1 regulator, as demonstrated in the Examples. For example, a portion of one of
the
above-described BRCA-1 regulators which has a sequence 5'-NS-GUC- Ng-3' or 5'-
NS-GUA- N8-3' is considered to be a TST. Such a TST can be used as a template
to
design novel ribozymes having RST sequences 5'-Ng-AGAA- N4-3' where Ng and
15 N4 have nucleotide sequences substantially complementary to the
corresponding
residues in the 5'-NS-GUC- N8-3' or 5'-NS-GUA- Ng-3' TST sequences. The RST
sequence can have between 8-12 nucleotides, and preferably 9-10 nucleotides at
positions Ng and N4 that are complementary to the corresponding 5'-NS-GUC- N8-
3'
or 5'-NS-GUA- Ng-3' TST sequences.
20 As described below in the Examples, additional ribozymes directed to a
variety of TST sequences on each of the previously described BRCA-1 regulators
have been constructed. Specifically constructed ribozymes were:
ribozymes selective for BBC1 having the RST sequences:
5'-GGCUUCAAAGAAAUGC-3', 5'-UGGGACCCAGAACGGG-3',
25 5'-CCGGAUGGAGAACGAC-3', 5'-ACGUUCCGAGAAGGCA-3',
5'-CCCAGCAUAGAAGCCC-3' (SEQ ID NOS: 22-26);
ribozymes selective for CHLR2 having the RST sequences:
5'-CCGAGAGAAGAAAGCC-3', 5'-UGGUUGGAAGAACCGA-3',
5'-GAGGAUGCAGAACCAC-3', 5'-AAGAAACAAGAAACCC-3',
5'-UUGGCCAGAGAAGGGG-3' (SEQ ID NOS: 27-31);
ribozymes selective for ID4 having the RST sequences:
5'-CAGUGGGCAGAACUCA-3', 5'-CCAACAAUUAGAAGGAG-3' ,
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5'-CACACCUGAGAAGCGC-3', 5'-CGCGGCUGAGAAGGUC-3'
(SEQ 1D NOS: 32-35);
ribozymes selective for AF6 having the RST sequences: 5'-
GUACUAGAAGAACGAA-3', 5'-UGUGAUCCAGAAAAGG-3', 5'-
GGUGGCCAAGAAGUGG-3' (SEQ ID NOS: 36-38);
ribozymes selective for BR2 having the RST sequences: 5'-
AAAAAUUAAGAAGUCA-3', 5'-GCUGUCCUAGAAUCAA-3', 5'-
UGUCAAAGAGAACACC-3', 5'-UGCAAUGAAGAAACUG-3',
5'-UUACAAUAAGAAACUU-3' (SEQ ID NOS: 39-43);
and ribozymes selective for BR3 having the RST sequences:
5'-CUAUUUAAAGAAAAUU-3', 5'-UAUUUCUUAGAAGUUC-3',
5'-AUUUCACUAGAAUCAC-3' (SEQ 1D NOS: 44-46).
Similarly, other types of methods can be used to corroborate the activity of a
BRCA-1 regulator nucleic acid containing at least about 8-12 nucleotides of a
TST
corresponding to SEQ 1D NO: 11-16. For example, an antibody or other selective
agent that binds a polypeptide encoded by the nucleic acid molecule can be
introduced into the cell, and the effect of the antibody on BRCA-1 expression-
or
activity determined, for example, by measuring the ability of the cells to
proliferate
in soft agar. Similarly, an antisense nucleic acid that inhibits transcription
or
translation of the BRCA-1 regulator nucleic acid can be introduced into a
cell, and
the effect of the antisense nucleic acid on BRCA-1 expression or activity
determined.
Likewise, an altered form of a BRCA-1 regulator nucleic acid molecule, such as
a
dominant-negative mutant, can be expressed in a cell and its encoded
polypeptide
will compete with or inhibit an endogenous BRCA-1 regulator molecule, and thus
increase BRCA-1 expression or activity. Those skilled in the art can determine
other
appropriate assays to demonstrate that a substantially pure nucleic acid
molecule
containing, at least about 8-12 nucleotides of any of SEQ >D NOS: 11-16 have
BRCA-1 regulator activity.
The TST sequences set forth as SEQ m NOS: 11-16, were identified from a
random hairpin ribozyme library by assessing the ability of their
corresponding RST
to increase BRCA-1 expression (SEQ ID NOS: 5-10). Therefore, the invention
provides ribozymes containing the RST sequences set forth as SEQ >D NOS: 5-10
as
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27
the ribozyme target recognition sequence. The hairpin ribozymes of the
invention
selectively bind to BRCA-1 regulator mRNA molecules complementary, in part, to
these RST sequences.
A substantially pure hairpin ribozyme of the invention can be catalytic, so as
to bind and cleave a BRCA-1 regulator nucleic acid messenger RNA. A catalytic
hairpin ribozyme of the invention can therefore be used to selectively
regulate the
activity of a BRCA-1 regulator nucleic acid molecule of the invention. A
substantially pure hairpin ribozyme of the invention can also be catalytically
disabled, for example, by replacement of the Loop 2 AAA sequence indicated in
Figure 1 with a UGC sequence, so as to bind, but not cleave, a BRCA-1
regulator
nucleic acid molecule of the invention. A non-catalytic hairpin ribozyme can
be
used, for example, as a control for the inhibition activity of non-disabled
ribozymes.
Therefore, the invention also provides a ribozyme containing a target
recognition sequence having any one of the following nucleotide sequences:
5'-CCGGAUGCAGAACAAU-3', 5'-AGUACAUUAGAAUACU-3' 5'
CUAGUGAGAGAAGGGA-3', 5'-UGAGAUCCAGAAAAGC-3'
5'-UGUUACUAGAAUGUU-3', and 5'-CCCUAUUUAGAAUUGU-3'
(SEQ m NOS: 5-10, respectively).
Also provided herein is a method of identifying ribozyme cleavage targets
from a nucleic acid sample by using the sequence specificity of the ribozyme
to
isolate a ribozyme cleavage product nucleic acid. This method can be used for
cloning a flanking sequence of any target nucleic acid for which there is a
ribozyme
cleavage site. A nucleic acid sample is subjected to cleavage by a sequence-
specific
test ribozyme. The sequence specific recognition and cleavage by a ribozyme
results
in at least one cleavage product nucleic acid having a specific sequence at
either the
5' or 3' end, which corresponds to the sequence recognized by the ribozyme.
When
the other end of the cleavage product nucleic acid contains a general
sequence, for
example, a 3' poly A tail, two primers can be constructed, a first
complementary to
the sequence recognized by the ribozyme, and a second complementary to the
general sequence. These two primers can then be used to amplify the cleavage
product nucleic acid, which then can be identified using known sequencing
methods.
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Thus from a nucleic acid sample, those nucleic acids that are cleaved by a
ribozyme
can be specifically amplified and subsequently identified.
Target nucleic acids can be either RNA or DNA, and can be cleaved by any
ribozyme, including a hairpin ribozyme, a hammerhead ribozyme and a
Tetrahymena
group I ribozyme. Prior to sequencing, amplification products can be separated
by
gel electrophoresis and can further be compared to a nucleic acid sample
subjected to
cleavage by a control ribozyme having sequence specificity different than the
ribozyme used in the nucleic acid sample being tested. The amplification
products
that are specific for the test ribozyme cleaved sample can be recovered and
sequenced.
One embodiment of this method uses four primers. A first primer hybridizes
to a 3' end first general sequence of the cleavage product nucleic acid, and
strand
synthesis is carried out. The enzyme used for such a synthesis places a second
general sequence at the 3' end of the newly synthesized strand, which also is
adjacent
the specific flanking sequence of ribozyme cleavage. Such an exemplary enzyme
is
MMLV reverse transcriptase, which places 3-5 deoxycytidine nucleotides at the
3'
end of the nascent DNA strand. Next, a second primer which contains a sequence
complementary to the second general sequence is added, and the nascent nucleic
acid
is further extended. The template strand is then removed, for example, by
RNAse
digestion if the template strand is RNA. The remaining nucleic acid is then
amplified using a third primer complementary to the sequence of the first
primer and
a fourth primer complementary to the sequence of the second primer and
containing
the flanking sequence of ribozyme cleavage. The amplified product is then
identified
using sequencing methods known in the art.
The nucleic acid molecules of the invention, including BRCA-1 regulator
nucleic acid molecules and fragments, and hairpin ribozyme nucleic acid
molecules,
can be produced or isolated by methods known in the art. The method chosen
will
depend, for example, on the type of nucleic acid molecule one intends to
isolate.
Those skilled in the art, based on knowledge of the nucleotide sequences
described
herein, can readily isolate BRCA-1 regulator nucleic acid molecules as genomic
DNA, or desired introns, exons or regulatory sequences therefrom; as full-
length
cDNA or desired fragments therefrom; or as full-length mRNA or desired
fragments
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therefrom, by methods known in the art. Likewise, those skilled in the art can
produce or isolate hairpin ribozymes selective for these sequences.
A useful method of isolating a BRCA-1 regulator nucleic acid molecule of
the invention involves amplification of the nucleic acid molecule using the
polymerase chain reaction (PCR), and purification of the resulting product by
gel
electrophoresis. For example, either PCR or reverse-transcription PCR (RT-PCR)
can be used to produce a BRCA-1 regulator nucleic acid molecule having any
desired
nucleotide boundaries. Desired modifications to the nucleic acid sequence can
also
be introduced by choosing an appropriate primer with one or more additions,
deletions or substitutions. Such nucleic acid molecules can be amplified
exponentially starting from as little as a single gene or mRNA copy, from any
cell,
tissue or species of interest. An example of the isolation of a BRCA-1
regulator
nucleic acid molecules using PCR are presented below in the Examples.
Another method of producing or isolating a BRCA-1 regulator nucleic acid
molecule of the invention is by screening a library, such as a genomic
library, cDNA
library or expression library, with a detectable agent. Such libraries are
commercially available or can be produced from any desired tissue, cell, or
species
of interest using methods known in the art. For example, a cDNA or genomic
library
can be screened by hybridization with a detectably labeled nucleic acid
molecule
having a nucleotide sequence disclosed herein. Additionally, an expression
library
can be screened with an antibody raised against a polypeptide corresponding to
the
coding sequence of a BRCA-1-regulator nucleic acid disclosed herein. The
library
clones containing BRCA-1 regulator nucleic acid molecules of the invention can
be
purified away from other clones by methods known in the art.
Furthermore, nucleic acid molecules of the invention can be produced by
synthetic means. For example, a single strand of a nucleic acid molecule can
be
chemically synthesized in one piece, or in several pieces, by automated
synthesis
methods known in the art. The complementary strand can likewise be synthesized
in
one or more pieces, and a double-stranded molecule made by annealing the
complementary strands. Direct synthesis is particularly advantageous for
producing
relatively short molecules, such as RST or hairpin ribozyme nucleic acid
molecules,
as well as hybridization probes and primers. Alternatively, nucleic acid
molecules
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of the invention can be produced using in vitro enzymatic synthesis using RNA
polymerase. For example, a template DNA can be combined with RNA polymerase
and ribonucleotide triphosphates. The RNA polymerase synthesizes an RNA
nucleic
acid complementary to the sequence of the DNA template.
5 If it is desired to subclone, amplify or express a substantially pure
nucleic
acid molecule of the invention, the isolated nucleic acid molecule can be
inserted
into a commercially available cloning or expression vector using methods known
in
the art. Appropriate regulatory elements can be chosen, if desired, to provide
for
constitutive, inducible or cell type-specific expression in a host cell of
choice, such
10 as a bacterial, yeast, amphibian, insect or mammalian cell, including human
cells.
Those skilled in the art can determine an appropriate host and vector system
for
cloning a nucleic acid molecule of the invention or for expressing and
purifying its
encoded polypeptide.
Methods for introducing a cloning or expression vector into a host cell are
15 well known in the art and include, for example, various methods of
transfection such
as the calcium phosphate, DEAF-dextran and lipofection methods, viral
transduction,
electroporation and microinjection. Host cells expressing BRCA-1 regulator
nucleic
acid molecules can be used, for example, as a source to isolate recombinantly
expressed BRCA-1 regulator polypeptides, to identify and isolate molecules
that
20 regulate or interact with BRCA-1 regulator nucleic acids and polypeptides,
or to
screen for compounds that enhance or inhibit the activity of a BRCA-1
regulator
molecule of the invention, as described further below.
The methods of isolating, cloning and expressing nucleic acid molecules of
the invention described herein are routine in the art and are described in
detail, for
25 example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold
Spring
Harbor Laboratory, New York (1992) and in Ansubel et al., Current Protocols in
Molecular Bioloay, John Wiley and Sons, Baltimore, MD (1989) .
BRCA-1 regulator polypeptides and functional fragments of the invention
can be isolated or prepared by methods known in the art, including
biochemical,
30 recombinant and synthetic methods. For example, a BRCA-1 regulator
polypeptide
can be purified by routine biochemical methods from a cell or tissue source
that
expresses abundant amounts of the corresponding transcript or polypeptide.
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Biochemical purification can include, for example, steps such as
solubilization of
the appropriate tissue or cells, isolation of desired subcellular fractions,
size or
affinity chromatography, electrophoresis, and immunoaffinity procedures. The
methods and conditions for biochemical purification of a polypeptide of the
invention can be chosen by those skilled in the art, and purification
monitored, for
example, by an ELISA assay or a functional assay.
A fragment having any desired boundaries and modifications to a BRCA-1
regulator amino acid sequences can also be produced by recombinant methods.
Recombinant methods involve expressing a nucleic acid molecule encoding the
desired polypeptide or fragment in a host cell or cell extract, and isolating
the
recombinant polypeptide or fragment, such as by routine biochemical
purification
methods described above. To facilitate identification and purification of the
recombinant polypeptide, it can be desirable to insert or add, in-frame with
the
coding sequence, nucleic acid sequences that encode epitope tags,
polyhistidine tags,
glutathione-S-transferase (GST) domains, and similar affinity binding
sequences, or
sequences that direct expression of the polypeptide in the periplasm or direct
secretion. Methods for producing and expressing recombinant polypeptides in
vitro
and in prokaryotic and eukaryotic host cells are well known in the art.
Functional fragments of a BRCA-1 regulator polypeptide can also be
produced, for example, by enzymatic or chemical cleavage of the full-length
polypeptide. Methods for enzymatic and chemical cleavage and for purification
of
the resultant peptide fragments are well known in the art (see, for example,
Deutscher, Methods in Enzymolo~y, Vol. 182, "Guide to Protein Purification,"
San
Diego: Academic Press, Inc. (1990)), which is incorporated herein by
reference.
Furthermore, functional fragments of a BRCA-1 regulator polypeptide can be
produced by chemical synthesis. If desired, such as to optimize their
functional
activity, stability or bioavailability, such molecules can be modified to
include D-
stereoisomers, non-naturally occurring amino acids, and amino acid analogs and
mimetics. Examples of modified amino acids and their uses are presented in
Sawyer,
Peptide Based Drug Designn, ACS, Washington (1995) and Gross and Meienhofer,
The Peptides: Analysis, Synthesis, Biology, Academic Press, Inc., New York
(1983), both of which are incorporated herein by reference.
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A functional activity of a BRCA-1 regulator polypeptide or fragment of the
invention can be its ability to alter BRCA-1 expression or activity, for
example
increasing unrestricted cell growth, when expressed or introduced in a cell.
To
determine whether a given polypeptide or fragment has the ability to alter
BRCA-1
expression or activity, a polypeptide or fragment can be expressed in the cell
by
recombinant methods known in the art and the effect of the BRCA-1 regulator
can be
determined in vitro. Alternatively, expression of the BRCA-1 regulator can be
inhibited in vivo, including cell culture or animal models and BRCA-1
expression or
activity can be determined. Similarly, expression of the BRCA-1 regulator can
be
inhibited in vivo, including cell culture or animal models and the expression
of a
BRCA-1 promoter-linked reporter marker determined. An increase in cell
proliferation in soft agar or decrease in the expression of a BRCA-1 promoter-
linked
reporter marker indicates that the polypeptide or fragment is a BRCA-1
regulator of
the invention.
The invention provides a method of identifying a compound that modulates
the activity of a BRCA-1 regulator comprising contacting a BRCA-1 regulator
with a
target molecule responsive to the activity of the BRCA-1 regulator and the
test
compound. In this method, an increase or decrease in the activity of the BRCA-
1
regulator for the target molecule compared to the absence of the test compound
indicates that the compound modulates the activity of the BRCA-1 regulator. As
used herein, "a target molecule responsive to the activity of the BRCA-1
regulator"
means that the BRCA-1 regulator either binds or chemically modifies the a
target
molecule that exists in a cell. For example, if the BRCA-1 regulator has DNA
binding activity for use in, for example, transcriptional gene regulation, the
target
molecule responsive to this activity is a nucleic acid comprising a sequence
of
nucleotides recognized and bound by the particular BRCA-1 regulator. If the
BRCA-1 regulator has protein kinase activity, due for example to having a
serine/
threonine kinase domain, the target molecule responsive to this activity is a
protein
having an appropriate serine or threonine kinase recognition site. Likewise,
for
activity as a protease, the target molecule responsive is a protein cleaved by
the
BRCA-1 regulator.
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When the BRCA-1 regulator activity to be measured for drug screening is
DNA binding, such binding can be determined by assaying the expression of a
reporter gene that is operatively linked to the nucleic acid element. In this
case, an
increase in the amount of expression or activity of the reporter gene in the
presence
of a test compound compared to the absence of the test compound indicates that
the
compound has BRCA-1 regulator DNA binding inhibitory activity. The magnitude
of the increase in expression activity will correlate with the BRCA-1
regulator
inhibitory activity of the test compound. Exemplary reporter genes include
BRCA-1,
EGFP and hygromycin resistance gene.
As used herein, the term "nucleic acid element" when used in reference to
regulation of BRCA-1 expression refers to a nucleic acid region that modulates
BRCA-1 expression. Exemplary nucleic acid elements are the BRCA-1 5' promoter
and regulatory region or other transcriptional regulation regions, and
translational
regulatory regions of the transcribed mRNA. Generally, the nucleic acid
element
will be the 5' promoter and regulatory region.
Similarly, compounds that increase or enhance the activity of BRCA-1
regulator also can be identified. A test compound added to a sample containing
a
BRCA-1 regulator and a nucleic acid element modulated by a BRCA-1 regulator
which decreases BRCA-1 activity or the amount or rate of expression of BRCA-1
or
a reporter gene operatively linked to the nucleic acid element compared to the
absence of the test compound indicates that the compound increases the
activity of
the BRCA-1 regulator. Therefore, the invention provides a method of
identifying
compounds that modulate the activity of a BRCA-1 regulator.
A reaction system for identifying a compound that inhibits or increases
BRCA-1 regulator activity can be prepared using essentially any sample,
material or
components thereof that contains a BRCA-1 regulator. A BRCA-1 regulator
containing sample used for such methods can be, for example, in vitro
transcription
or translation systems using, for example, nucleic acid derived from the BRCA-
1
gene-of a normal or tumor cell or a hybrid construct linking the nucleic acid
element
modulated by a BRCA-1 regulator to a reporter gene. Alternatively, nucleic
acids
and proteins obtained from normal cells can also be used since BRCA-1
regulators
can also act in normal cells. The BRCA-1 regulator-containing sample can
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34
additionally be derived from cell extracts, cell fractions or, for example, in
vivo .
systems such as cell culture or animal models which contain a nucleic acid
element
modulated by a BRCA-1 regulator. The expression levels or activity of BRCA-1
or
the reporter gene can be measured in the reaction system to determine the
modulatory effect of the test compound on the BRCA-1 regulator. Such
measurements can be determined using methods described herein as well as
methods
well known to those skilled in the art.
Briefly, the BRCA-1 regulator source is combined with a nucleic acid
element or protein modulated by a BRCA-1 regulator as described above and
incubated in the presence or absence of a test compound. The expression levels
or
activity of BRCA-1 or the reporter gene in the presence of the test compound
is
compared with that in the absence of the test compound. Those test compounds
which provide an increase in expression levels or activity of BRCA-1 or the
reporter
gene of at least about 50% are considered to be BRCA-1 regulator inhibitors,
or
antagonists, and further, potential therapeutic compounds for the treatment of
neoplastic diseases such as cancer. Similarly, those compounds which decrease
expression levels or activity of BRCA-1 or the reporter gene by about two-fold
or
more are considered to be compounds which increase the activity of a BRCA-1
regulator, or BRCA-1 regulator agonists. Such agonists can be used as
therapeutics,
for example, to promote cell growth or cell survival in transplanted or
explanted cells
which are subsequently transplanted. Compounds identified to modulate BRCA-1
regulator activity can, if desired, be subjected to further in vitro or in
vivo studies to
corroborate that they affect the activity of a BRCA-1 regulator toward the
BRCA-1
expression or activity.
Suitable test compounds for the above-described assays can be any substance,
molecule, compound, mixture of molecules or compounds, or any other
composition
which is suspected of being capable of inhibiting BRCA-1 regulator activity in
vivo
or in vitro, for example, compounds with cell proliferation-inhibiting
activity. The
test compounds can be macromolecules, such as biological polymers, including
proteins, polysaccharides and nucleic acids. Sources of test compounds which
can be
screened for BRCA-1 regulator inhibitory activity include, for example,
libraries of
small organic molecules, peptides, polypeptides, DNA, and RNA. Additionally,
test
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compounds can be pre-selected based on a variety of criteria. For example,
suitable
test compounds can be selected as having known inhibition or enhancement
activity
with respect to cell proliferation. Alternatively, the test compounds can be
selected
randomly and tested by the screening methods of the present invention. Test
5 compounds can be administered to the reaction system at a single
concentration or,
alternatively, at a range of concentrations to determine, for example, the
optimal
modulatory activity toward the BRCA-1 regulator.
The activity of a BRCA-1 regulator for which drug screening is desired can
be a protein kinase activity. For example, BRCA-1 regulators that have a
10 serine/threonine kinase domain may be used for drug screening where the
activity
which is modulated is a protein kinase activity. Protein kinase assays are
well
known to those skilled in the art (see, e.g., U.S. Pat. Nos. 5,538,858 and
5,757,787;
Anal. Biochem, 209:348-353, (1993)).
The activity of a BRCA-1 regulator for which drug screening is desired also
15 can be GTP binding activity. For example, BRCA-1 regulators that have a GTP
binding site may be used for drug screening where the activity which is
modulated is
GTP binding. BRCA-1 regulators that have GTP-binding activity may regulate
cell
growth such as through regulating BRCA-1 expression, or may have affects on
cell
cycle control, protein secretion, and intracellular vesicle interaction. GTP
binding
20 assays are well known to those skilled in the art (see, e.g., U.S. Pat.
Nos. 5,840,969
to Hillman et al.).
The activity of a BRCA-1 regulator for which drug screening is desired also
can be hormone binding activity. For example, BRCA-1 regulators that have a
hormone binding site may be used for drug screening where the activity which
is
25 modulated is hormone binding. Hormones that bind to a BRCA-1 regulator may
be
steroid hormones such as estrogen or a protein based hormone. Receptor hormone
binding assays including receptor estrogen binding assays are well known to
those
skilled in the art (see, e.g., U.S. Pat. Nos. 6,204,067 to Simon et al.).
The invention provides a method of identifying a ribozyme reactive with a
30 BRCA-1 regulator. The method comprises: (a) introducing a randomized
ribozyme
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36
library into a population of cells expressing a selection marker gene
operatively
linked to a nucleic acid element modulated by a BRCA-1 regulator; (b)
subjecting
the population of cells to selection, and (c) recovering one or more ribozymes
from
viable cells following selection.
Also provided is a method of identifying a BRCA-1 regulator. The method
comprises: (a) introducing a randomized ribozyme library into a population of
cells
expressing a selection marker operatively linked to a nucleic acid element
modulated
by a BRCA-1-regulator; (b) subjecting the population of cells to selection;
(c)
recovering one or more ribozymes from viable cells following selection; and
(d)
sequencing the target recognition sequence of the recovered ribozyme to
identify the
nucleic acid encoding the BRCA-1 regulator.
By reference to cancer, or to breast or ovarian cancer in particular, as an
exemplary neoplastic disease amenable to the methods of identifying a ribozyme
or a
BRCA-1 regulator of the invention, one skilled in the art will readily know,
in light of
the teachings and description herein that such methods are applicable to
essentially
all neoplastic diseases which employ tumor suppressor regulators for continued
propagation. Therefore, the methods of identifying a BRCA-1 regulator, or
ribozyme
selective to a BRCA-1 regulator, as well as methods of treating a neoplastic
disease
once such regulators have been identified are applicable to methods of
identifying
any tumor suppressor regulator and to methods of treating a neoplastic
disease.
A method of identifying tumor suppressor regulators can be carried out using
the methods described herein. Briefly, a nucleic acid element of a tumor
suppressor
gene that regulates the expression of the gene, for example, the 5' promoter
and
regulatory region for a specific tumor suppressor, can be operatively linked
to a
reporter gene. A sample containing a tumor suppressor regulator and a nucleic
acid
element modulated by the tumor suppressor regulator linked to a reporter gene
can
be contacted with a test compound and the expression or activity of the
reporter gene
can be measured. A test compound found to increase reporter gene expression or
activity indicates that the test compound inhibits tumor suppressor regulator
activity.
A test compound found to decrease reporter gene expression or activity
indicates that
the test compound increases or enhances tumor suppressor regulator activity.
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For the successful application of such methods, it is sufficient to have
identified a nucleic acid, derived from a normal or tumor cell, which is
modulated by
a tumor suppress or regulator, for example, the BRCA-1 promoter or the p53
promoter. Once identified, the nucleic acid derived from the cell can be
operatively
linked to a reporter gene such as a selection marker gene, and subjected to
selection
using the methods of the invention.
Moreover, it is not necessary for the nucleic acid derived from the normal or
tumor cell to be unique to the regulation of expression or activity of the
specific
tumor suppressor in that cell. Instead, the normal or tumor cell-derived tumor
suppressor regulator nucleic acid can overlap or be redundant with other
regulators
of tumor suppressor expression or activity. The expression or activity level
of the
tumor suppressor can rely on a balance between levels of one or more tumor
suppressor regulators found in normal cells compared to levels of one or more
tumor
suppressor regulators in tumor cells. Therefore, decreasing the level or
activity of
tumor suppressor regulators acting on common components or structures can
shift
the balance toward utilization of tumor suppressor regulators for normal
cellular
functions reducing the amount of unrestricted cell growth. An example of a
nucleic
acid modulated by a tumor suppressor regulator includes a nucleic acid that is
expressed in normal cells, but whose expression is increased in tumor cells.
Another
example of a nucleic acid modulated by a tumor suppressor regulator includes a
nucleic acid that is not expressed in normal cells, but is expressed in tumor
cells.
The method of identifying a ribozyme reactive with a BRCA-1 regulator
involves the construction of a population of cells expressing a selection
marker gene
which is under the control, or operatively linked to a nucleic acid modulated
by a
BRCA-1 regulator. Specific examples of such a cell population and its use are
described further below in the Examples.
Briefly, a nucleic acid element modulated by a BRCA-1 regulator can be
essentially any nucleic acid sequence that influences BRCA-1 expression or
activity.
Specific examples of such an element includes the BRCA-1 promoter and the 5'
regulator region. Methods using BRCA-1 regulators identified for the BRCA-1
promoter and therapeutic compounds directed thereto are applicable to all
neoplastic
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38
diseases related to decreased BRCA-1 expression or activity. Other elements
can
include, for example, transcription enhancers and BRCA-1 mRNA.
Cell populations containing a nucleic acid element modulated by a BRCA-1
regulator are operatively linked to a selection marker gene. Operative linkage
will
depend on the type of element employed and is intended to refer to placing the
nucleic acid element in an appropriate context and location in the reporter
construct
as it would be found in its native genome. In the specific example of BRCA-1
promoter, operative linkage places the element 5' to the transcription start
site of the
marker gene. Operative linkage of a promoter element-will be sufficiently
upstream
of the translation start codon to include sufficient 5' untranslated region
sequence to
effect translation in, for example, a CAP-dependent manner. The reporter
constructs
can be introduced into cell population using well known methods in the art and
as
described previously.
A selection marker can be a gene product that is, or can be made to influence
cell viability or cell growth, or can be used as a measurable indicator.
Specific
examples include enhanced green fluorescence protein (EGFP), hygromycin
resistance gene, the tumor suppressor BRCA-1, Herpes Simplex Virus thymidine
kinase (HSV-tk), cytosine deaminase (CD) and diphtheria toxin (DT). For
example,
the expression of a negative selection marker in cells is either toxic alone,
or toxic in
the presence of a negative selection compound which is metabolized by the
marker
gene product into a cytotoxic or cytostatic substance. In contrast, expression
of a
positive selection marker in cells protects cells from arrested growth or cell
death.
Alternatively, the gene product can be a measurable indicator that can be used
in
selecting cells having a certain measured level of expression of this gene
product.
Expression of an easily detectable gene product such as EGFP can be used as
a measurable indicator of gene expression. EGFP allows a specific, user-
defined
expression level to be selected using methods such as fluorescence activated
cell
sorting (FAGS). For example, cells representing the 10°Io of the
population
expressing the highest levels of EGFP can be recovered using FACS methods. The
selected subpopulation can be grown and subjected to a second round of FACS-
based selection of high level EGFP expression. This iterative FACS-based
selection
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39
results in enrichment of cells expressing high levels of EGFP and thus
represents a
FACS-based positive selection method.
Positive selection can also be carned out, for example, using hygromycin.
Expression of the hygromycin resistance gene permits cells to survive in the
presence
of hygromycin. Hygromycin is an aminoglycoside antibiotic that kills bacteria,
fungi
and higher eukaryotic cells by inhibiting protein synthesis. Expression of
hygromycin resistance gene allows cells to survive in the presence of
hygromycin.
Concentrations of hygromycin used for selection are between about 50 pg/ml and
1000 pg/ml, preferably about 250 pg/ml.
Phenotype selection can also be carried out. Expression of the BRCA-1 gene
suppresses cell growth, and particularly anchorage-independent growth,
resulting in,
for example, a decreased ability to grow in soft agar. Growth of cells in soft
agar can
therefore be used to determine the propensity of the cells for anchorage-
independent
growth, thus indicating the level of BRCA-1 gene expressed.
Once selection proceeds, the selected cells are those which express a
ribozyme that is reactive with a BRCA-1 regulator that inhibits or decreases
BRCA-1
promoter-dependent expression of the reporter gene. The cells are isolated and
the
ribozymes are recovered using, for example, PCR or other well known methods in
the art. The RST of the ribozyme is a sequence tag corresponding to a BRCA-1
regulator. Sequencing of this tag identifies the nucleic acid encoding the
BRCA-1
regulator. Specific examples of RSTs corresponding to BRCA-1 regulators of the
invention are set forth as SEQ )D NOS: 5-10 and their corresponding TSTs are
set
forth as SEQ >D NOS: 11-16. Five RST sequences have been determined.
Specifically, SEQ ID NO: 7 corresponds to the RST for BR1, the full length
nucleotide and amino acid sequences of which is shown as SEQ >D NOS: 1 and 2.
SEQ >D NO: 5 corresponds to the RST for BBC1 (GenBank accession number
X64707) and also for CHLR2 (GenBank accession number U33834) and SEQ )D
NO: 8 corresponds to the RST for >D4 (GenBank accession number NM_001546)
and also for AF6 (GenBank accession number AB011399). SEQ ID NO: 6
corresponds to the RST for BR2 (GenBank accession number AL045940), and SEQ
)17 NO: 9 corresponds to the RST for BR3 (GenBank accession number AI276397).
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The invention also provides a method of treating breast cancer or ovarian
cancer. The method consists of introducing a ribozyme selectively reactive
with a
RNA encoding BBC1, BR1, ID4, BR2, or BR3, into a cancerous breast or ovarian
cell, preferably the ribozyme is selectively reactive with an RNA encoding
BBC1,
5 >D4 or BR1. Also provided is a method of treating breast or ovarian cancer
by
introducing a ribozyme selectively reactive with a RNA encoding a BRCA-1
regulator corresponding to a RST selected from the group consisting of SEQ m
NOS: 5-10. By substituting the ribozymes of the invention selectively reactive
with
a BRCA-1 regulator RNA with an antisense nucleic acid corresponding to a RST
10 sequence selected from the group consisting of SEQ m NOS: 5-10, methods of
treating breast cancer or ovarian cancer are also provided. The antisense
nucleic
acids hybridize to the BRCA-1 regulator nucleic acid similar to catalytic
ribozymes
and inhibit transcription processing or translation of the RNA without
subsequent
cleavage. Such methods will be described below with reference to a ribozyme of
the
15 invention, but those skilled in the art will know that antisense nucleic
acids can
similarly be substituted for the ribozymes to prevent or reduce the severity
of breast
cancer or ovarian cancer.
A ribozyme encoding any of the RST sequences set forth as SEQ >D NOS: 5-
10, or a combination thereof can be delivered in a wide variety of ways to
tumor
20 cells or cells susceptible to uncontrolled proliferation to interrupt or
prevent
neoplastic growth. The ribozyme can be administered as RNA or expressed from
an
expression vector. The ribozyme can be administered ex vivo by, for example,
administering to cells that have been removed from an infected individual, and
then
returned to the individual, or the ribozyme can be administered in vivo.
Delivery can
25 be performed using any appropriate delivery vehicle known to those skilled
in the art
including, for example, a liposome, a controlled release vehicle,
electroporation or
covalently attached moieties, and other pharmacologically acceptable methods
of
delivery. A carrier can provide specificity for tissue or organ accumulation,
such as
breast or ovary accumulation, of the ribozyme at the tissue or organ which is
the
30 primary site of the neoplastic-growth. The ribozyme delivery vehicle can be
designed
to serve as a slow release reservoir or to deliver its contents directly to
the target cell.
Examples of ribozyme delivery vehicles include liposomes, hydrogels,
cyclodextrins,
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41
biodegradable nanocapsules, and bioadhesive microspheres. Liposomes can
readily
be targeted to the liver for delivery of RNA to infected tumor cells.
Routes of ribozyme administration include intramuscular, aerosol,
intravenous, parenteral, intraperitoneal. Generally however, the route of
administration will be through a blood vessel which represents a direct route-
to the
organ or tissue to be treated. The dosage of ribozyme will also depend on a
variety of
factors, such as the form of the ribozyme, the route of administration, the
severity of
the cancer or stage of disease, the general condition of the patient being
treated, and
thus can vary widely. Generally the dosage of ribozyme will be between about
lOpg
- 200mg/kg of body weight per day and result in therapeutic or prophylactic
levels
within the targeted cells sufficient to inhibit or eradicate the tumor cells.
The
duration of treatment can extend throughout the course of the neoplastic
disease or
disease symptoms, usually at least about 7-30 days, with longer duration being
necessary for severe diseases. The number and timing of doses can also vary
depending on, for example, the extent of disease.
A viral vector containing a ribozyme corresponding to a BRCA-1 regulator
RST of the invention can be prepared in any of a wide variety of ways known to
those skilled in the art. Representative retroviral vectors which can be used
in the
methods of the invention are described, for example, in U.S. Patent Nos.
4,861,719,
5,124,263 and 5,219,740. Other vectors can also be employed, particularly for
the ex
vivo methods, such as DNA vectors, pseudotype retroviral vectors, adenovirus,
and
adeno-associated virus vectors.
The viral vector contemplated for use in the methods of the present invention
comprises infectious, but replication-defective, viral particles, which
contain at least
one DNA sequence encoding a ribozyme selectively reactive with a BRCA-1
regulator, is administered in an amount effective to inhibit or prevent cancer
formation or progression in a subject. The vector particles can be
administered in an
amount-from 1 plaque forming unit to about 1014 plaque forming units, more
preferably from about 1X106 plaque forming units to about 1X1013 plaque
forming
units. A sufficient number of vector particles containing a ribozyme
selectively
reactive with a BRCA-1 regulator of the invention is administered to the
cancerous
tissue or organ to infect up to at least about 50°7o of the cancerous
cells, usually about
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42
80%, preferably about 90%, or more of the cancerous cells in the individual.
Subsequent administrations can be performed, as needed, to effectively treat
or
reduce the severity of the cancer.
Exemplary ribozymes of the invention include, for example, those having
RST sequences set forth as SEQ ID NOS: 5-10. One of these RST sequences, SEQ
ID NO: 5, corresponds to the proteins BBC1 and CHRL2. Another RST sequence,
SEQ ID NO: 8, corresponds to the proteins ID4 and AF6. Three of the RST
sequences correspond to sequences without previously known roles. Briefly, SEQ
>D
NO: 7 corresponds to BR1 (SEQ >D NO: 1), SEQ >D NO: 6 corresponds to BR2 and
SEQ ID NO: 9 corresponds to BR3.
Methods of treating cancer are also provided by this invention. More
specifically, within one aspect of the present invention cancerous conditions
may be
treated by administering to a warm-blooded animal (e.g., a human) a
therapeutically
effective amount of ribozyme.
The present invention provides for treatment of cancer by contacting desired
cells with an effective amount of ribozyme of this invention. A suitable
"therapeutically effective amount" will depend on the nature and extent of
diseased
tissue being treated. Such "therapeutically effective amounts" can be readily
determined by those of skill in the art using well known methodology, and
suitable
animal models such as a rat, rabbit, or porcine model, or, based upon clinical
trials.
As utilized herein, a patient is deemed to be "treated" if the cancerous
condition is
reversed or inhibited in a patient in a quantifiable manner. A therapeutically
effective
amount or regimen of treatment should result in: (1) decrease in the
frequency,
severity, or, duration of clinical symptoms (e.g., pain); (2) increase of time
in the
period of remission; (3) a change in pathological symptoms (e.g. reduction in
tumor
volume or tumor markers); or (4) any combination thereof. When exogenously
delivering the ribozyme, the RNA molecule can be embedded within a stable RNA
molecule or in another form of protective environment, such as a liposome.
Alternatively, the RNA can be embedded within Rnase-resistant DNA
counterparts.
Cellular uptake of the exogenous ribozyme can be enhanced by attaching
chemical
groups to the DNA ends, such as cholesteryl moieties (Letsinger et al., Proc.
Natl.
Acad. Sci. USA, 1989).
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Hammerhead ribozymes suitable for use within the present invention
preferably recognize the sequence NUH, wherein N is any of G, U, C, or A and H
is
C, U, or A. The hairpin recognition sites GUC are a subset of the hammerhead
recognition sites NUH. Therefore, all hairpin sites are by definition also
hammerhead
sites, although the converse is not true. Chimeric hammerhead ribozymes (i.e.,
RNA/DNA hybrids) are designed to have an appropriate NUH sequence for
ribozyme cleavage. Ribozymes are chemically synthesized with the general
structure
shown below as Scheme 1. The binding arms bases and stem loop comprise DNA,
and the catalytic domain comprises RNA and/or 2' O methyl RNA bases. The stem
loop can be replaced by a propanediol linker. DNA bases are shown in upper
case,
RNA bases in lower case, 2' O methyl RNA is shown as lower case underlined,
and
propanediol linker is shown as pr pr pr pr.
Scheme 1
Sequence ID No: 47 Length: 38
5' NNNNN nn cuga a g ~ CCGTAAGG cc ga a a cc NNNNNN 3'
Sequence ID No: 48 Length: 28
5' I\fNNNN nn cuga a g ~pr pr pr pr c ga a a cc NNNNNN 3'
In addition to the methods of treating breast cancer or ovarian cancer using
ribozymes of the invention, inhibitory compounds identified by the screening
methods described previously can similarly be used to reduce the severity of
breast
cancer or ovarian cancer. Small organic compounds have particular advantages
because of their ease of formulation and administration using well known
methods in
the pharmaceutical arts.
The present invention provides methods to identify gene targets that are down
regulated by ribozyme activity. Another powerful application of this
technology is
to identify other genes in the pathway that contribute to the phenotype
changes
observed after ribozyme knockdown. Expression profiles can be determined in
hybridization arrays of cDNA targets from known genes or expressed sequence
tag
(EST) sequence databases.
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In one embodiment, differentially expressed mRNA in control ribozyme and
effector ribozyme transduced cells, can be detected by reverse transcribing
the
mRNA from the cells into cDNA, which is labeled with green (control ribozyme)
and red fluorescent dye (effector ribozyme). Individual EST clones are arrayed
on a
solid support such as polylysine-coated glass (e.g., a 24 x 24 panel). The
microarray
is hybridized with a mix of the two labeled cDNA and then scanned for
fluorescent
color. A G3pDH DNA fragment is spotted on the corners of each 24x24 panel and
serves as an internal control to check for an equal amount of cDNA applied
from the
two cell lines. The intensity of the color at each location of the array is
proportional
to the expression level of that gene in the sample. EST fragments which showed
a
predominant red color indicate that the EST is expressed mostly in the
effector
ribozyme cells or up-regulated by effector ribozyme treatment, while a
predominance of green color indicates that the EST is expressed largely in the
control ribozyme cells or down-regulated by effector ribozyme treatment.
In the above approach, arrays of clones can be formed on any of a variety of
solid supports, including, for example a membranes such as made from nylon or
a
silicon based chip. Also, the cDNA prepared from the two transduced cell types
may
be differentially labeled by fluorophores, enzymes, radioisotopes and the
like. If
radiolabeled cDNA is used, each pool of labeled cDNA is hybridized to separate
membranes, which are scanned for radioactive intensity using a phosphorimager.
The radioactive intensity of each array element is proportional to the number
of
bound cDNA molecules, so the intensities of effector ribozyme cDNA can be
directly compared to control ribozyme cDNA in a similar fashion to the
fluorescent
elements described above.
Criteria such as at least a 2 fold difference in expression level can be set
to
delineate potential targets. for genes involved in the pathway leading to the
phenotypic change. Using this technology, additional drug targets indirectly
regulated by the ribozyme can be identified.
Regulators of BRCA-1 expression were identified using the above expression
profiling approach as described in greater detail in Example X. These genes,
which
included GenBank Acc. No. AA419229, GenBank Acc. No. H18950, GenBank Acc.
No. H07920, GenBank Acc. No. H70047, GenBank Acc. No. H84815, GenBank
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Acc. No. AA757764, GenBank Acc. No. H19111, GenBank Acc. No. AA629897,
GenBank Acc. No. AA663439, GenBank Acc. No. 815740, GenBank Acc. No.
AA454570, GenBank Acc. No. AA485748, GenBank Acc. No. AA464601,
GenBank Acc. No. AA102107: and GenBank Acc. No. 249826,
5 represent therapeutic drug or ribozyme effector targets for increasing BRCA-
1
expression.
The invention also provides a high throughput drug discovery method using
ribozyme transduced cells and chip array technology. By combining array
technology with ribozyme knockdown, drugs can be rapidly screened for effects
on a
10 given pathway. Once the expression profile leading to a given phenotype is
determined, additional arrays can be generated with the relevant regulated EST
sequences. These can be screened with mRNA from drug treated cells. Profiles
matching the ribozyme treated profile can be identified. Treatment with the
drugs
identified in this way can be expected to give the desired phenotype.
15 This methodology allows the linking of the function of these target genes
to
the desired phenotype i.e., increased BRCA-1 expression. Small molecule drugs,
ribozyme drugs, or antibody drugs can be identified by those skilled in the
art that
inhibit the activity of these gene targets resulting, for example, in
increased BRCA-1
expression. The gene targets can be used to develop high throughput assays
that can
20 be screened with existing small molecule libraries. In addition, genes
which express
a surface or secreted protein can be targets for antibody development.
Antibodies
specific for the gene product can be generated preferably in transgenic mouse
systems to generate human antibodies. Furthermore, chimeric ribozyme drugs
targeting these BRCA-1 regulators can be designed as explained above (see
e.g.,
25 scheme 1 above and scheme 2 below).
The invention also provides a method of detecting a neoplastic cell in a
sample. In one embodiment, the method consists of contacting the sample with a
detectable agent specific for a BRCA-1 regulator nucleic acid molecule, and
detecting the nucleic acid molecule in the sample. Altered expression or
structure of
30 the nucleic acid molecule indicates the presence of a neoplastic cell in
the sample. In
another embodiment, the method consists of contacting the sample with a
detectable
agent specific for a BRCA-1 regulator polypeptide of the invention, and
detecting the
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46
polypeptide in the sample. Altered expression or structure of the polypeptide
indicates the presence of a neoplastic cell in the sample.
The diagnostic methods described herein are applicable to the identification
of neoplastic cells present. in solid tumors (carcinomas and sarcomas) such
as, for
5. example, breast cancer, ovarian cancer and prostate cancer. The BRCA-1
regulator
molecules that can be detected using this method include nucleic acids
encoding
BR1, BR2, BR3, BBC1, or ID4 or the corresponding polypeptides.
Various qualitative and quantitative assays to detect altered expression or
structure of a nucleic acid molecule in a sample are well known in the art,
and
generally involve hybridization of a detectable agent, such as a complementary
primer or probe, to the nucleic acid molecule. Such assays include, for
example, in
situ hybridization, which can be used to detect altered chromosomal location
of the
nucleic acid molecule, altered gene copy number, or altered RNA abundance,
depending on the format used. Other assays include, for example, RNA blots and
RNase protection assays, which can be used to determine the abundance and
integrity of RNA; DNA blots, which can be used to determine the copy number
and
integrity of DNA; SSCP analysis, which can detect single point mutations in
DNA,
such as in a PCR or RT-PCR product; and coupled PCR, transcription and
translation
assays, such as the Protein Truncation Test, in which a mutation in DNA is
determined by an altered protein product on an electrophoresis gel. Further
assays
include methods known in the art for genotyping a BRCA-1 regulator gene, for
example, by RFLP analysis or by determining specific SNPs in a BRCA-1
regulator
gene. An appropriate assay format and detectable agent to detect an alteration
in the
expression or structure of a BRCA-1 regulator nucleic acid molecule can be
determined by one skilled in the art depending on the alteration one wishes to
identify.
Various assays to detect altered expression or structure of a BRCA-1
polypeptide are also well known in the art, and generally involve
hybridization of a
detectable agent, such as an antibody or selective binding agent, to the
polypeptide in
a sample. Such assays can be performed in situ, such as by
immunohistochemistry or
immunofluorescence, in which a detectably labeled antibody contacts a
polypeptide
in a cell. Other assays, for example, ELISA assays, immunoprecipitation, and
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immunoblot analysis, can be performed with cell or tissue extracts. Assays in
which
the polypeptide remains in a native form are particularly useful if a
conformation-
specific binding agent is used, which can detect a polypeptide with an altered
structure. A structural variant of a BRCA-1 regulator polypeptide can act, for
example, in a dominant-negative fashion to decrease BRCA-1 expression or
activity
and cause unregulated cell proliferation. An appropriate assay format and
detectable
agent to detect an alteration in a BRCA-1 regulator polypeptide can be
determined
by one skilled in the art depending on the alteration one wishes to identify.
Also contemplated herein are in vivo methods for detecting a BRCA-1
polypeptide or nucleic acid, comprising administering to a subject a
detectable agent
such as an antibody or a complementary RNA molecule, appropriately conjugated
with an imaging reagent useful for in vivo detection of the detectable
compound
using methods known in the art such as magnetic resonance imaging, computed
tomography, x-ray imaging, and the like. Imaging reagents are well known in
the art
and include radioactive reagents, electron-dense reagents and magnetic or
electronic
reagents.
The diagnostic methods described herein can also be used in prognostic
assays. Such an application can identify alterations in expression or
structure of
BRCA-1 regulator molecules that take place at characteristic stages in the
progression of a proliferative disease or of a tumor. Knowledge of the stage
of the
tumor allows the clinician to select the most appropriate treatment for the
tumor and
to predict the likelihood of success of-that treatment. The diagnostic methods
described herein can also be used to monitor the effectiveness of therapy.
Successful
therapy can be indicated, for example, by a reduction in the number of
neoplastic
cells in an individual, as evidenced by more normal expression or structure of
the
BRCA-1 regulator in a sample following treatment.
In the diagnostic and prognostic assays described herein, the level of
expression or structure of the detected BRCA-1 regulator nucleic acid or
polypeptide
in the test sample is compared to the known expression level or structure of
the
nucleic acid or polypeptide in a normal sample. The normal sample can be
obtained
either from normal tissue of the same histological origin of the same or a
different
individual, or a population of individuals.
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The diagnostic methods described herein can also be used in a method for
determining the propensity of a subject to develop cancer. Such a method
includes
collecting a pre-neoplastic sample from a subject which can include a sample
of a
specific tissue or organ in which a neoplastic growth is suspected to occur,
or
alternatively a sample from any portion of the subject, such as a blood,
saliva or
cheek swab sample. The detected expression level or structure of a BRCA-1
regulator nucleic acid or polypeptide is then compared to the corresponding
expression level or structure known for a population of normal and cancer
subjects,
whereby the expression level or structure will correspond to a specific
subpopulation
of subjects. The number of normal subjects relative to cancer subjects in the
specific
subpopulation can be determined and results in a likelihood value of any
subject
within that subpopulation for developing cancer. The propensity of the subject
to
developing cancer is determined by assigning to it the likelihood value of the
subpopulation to which the expression level or structure of the subject
corresponds.
It is understood that modifications which do not substantially affect the
activity of the various embodiments of this invention are also included within
the
description of the invention provided herein. Accordingly, the following
examples
are intended to illustrate but not limit the present invention.
EXAMPLES
EXAMPLE I:
Preparation of the random retroviral vector ribozyme library
This example demonstrates the construction of a random retroviral vector
ribozyme gene library. The inventors have discovered that by introducing a
random
retroviral vector ribozyme gene library in the PA1 ovarian carcinoma cell
line,
certain of the ribozymes will selectively target and inactivate mRNA molecules
encoding regulators of the BRCA-1 tumor suppressor gene. A ribozyme gene
library
with randomized target recognition sequences was introduced into mammalian
cells
stably expressing a selectable marker, enhanced green fluorescent protein
(EGFP)
under the control of the BRCA-1 promoter. Cells in which BRCA-1 expression was
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upregulated by particular ribozymes were selected through their concomitant
increase in EGFP expression. The ribozyme genes were then rescued from the
cells
with enhanced EGFP expression and sequenced across their substrate binding
sites.
The corresponding ribozyme binding sequence, or "target sequence tag" (TST) is
a
sequence present in the regulator nucleic acid molecule targeted by the
ribozyme.
Thus, knowledge of the TST allows novel regulator nucleic acids to be
identified and
isolated.
A plasmid-based retroviral library was constructed by inserting random
ribozyme gene sequences into parent vector pLHPM-2kb. pLHPM-2kb contains 5'
and 3' long terminal repeats (LTR) of the Moloney retroviral genome; a
neomycin
resistance gene driven by the LTR; an SV40 promoter driving a puromycin
resistance gene; and a transcription cassette containing a tRNAvaI promoter
and a 2
kb stuffer insert. When the stuffer insert is removed and replaced by the
random
ribozyme library inserts, the tRNAvaI promoter can drive transcription of the
inserted ribozyme gene.
To prepare the pLHPM-2kb vector, plasmid pLHPM was digested overnight
at 65°C with BstBl, phenol: chloroform extracted and ethanol
precipitated. The
resuspended DNA was then digested overnight at 37°C with MIuI. This
double
digestion excises the 2kb stuffer fragment. The resultant 6kb plasmid vector
DNA
fragment was purified by agarose gel electrophoresis.
To prepare the random ribozyme library inserts, three oligonucleotides were
synthesized and annealed in annealing buffer (50 mM NaCI, 10 mM Tris pH 7.5, 5
mM MgCl2) at a molar ratio of 1:3:3 (oligol:oligo2:oligo3) by heating to
90°C
followed by slow cooling to room temperature. The three oligonucleotides had
the
following sequences:
Oligol: 5'-pCGCGTACCAGGTAATATACCACGGACCGAAGTCCG
TGTGTTTCTCTGGTNNNNTTCTNNNNNNNNGGATCCTGTTTCCGCC
CGGTTT-3'
(SEQ ~ NO: 49)
Oligo2: 5'-pGTCCGTGGTATATTACCTGGTA-3' (SEQ ID NO: 50)
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Oligo3: 5'-pCGAAACCGGGCGGAAACAGG-3' (SEQ ID NO: 51)
To provide for random and uniform incorporation of A, T, C and G
nucleotides at the positions represented as N nucleotides in oligol, the A, T,
C and G
reagents were premixed, and the same mixture used for every N position in the
oligonucleotide synthesis. The ribozyme insert library formed by annealing the
three
oligonucleotides (SEQ ID NOS: 7-9) thus contains 8 positions with random
nucleotides corresponding to helix 1 of the ribozyme, and 4 random positions
with
10 random nucleotides corresponding to helix 2 of the ribozyme.
In order to ligate the pLHPM-2kb vector DNA fragment with the random
ribozyme insert library, 0.5 pmole of the vector and an 8-fold molar excess of
annealed oligonucleotides were ligated overnight with 10 units of T4 DNA
ligase.
Ultracompetent DH12S bacteria were then electroporated with the ligation
mixture.
15 A total of 5 x 10' bacterial colonies containing the retroviral plasmid
ribozyme
library was obtained.
The bacterial colonies containing the retroviral plasmid ribozyme library
were pooled in aliquots as a master stock and frozen at -80°C. Working
stocks were
made by culturing 1 ml of the master stock in 60 ml LB media overnight at
30°C. A 1
20 ml aliquot of the working stock was used to make a 500 ml bacterial culture
by
incubation at 30°C overnight. Plasmid DNA was then-extracted from the
500 ml
culture and used to generate retroviral vector as described below.
Following the cloning of the randomized hairpin ribozyme genes into
pLHPM, the "randomness" of the plasmid library was evaluated by both
statistical
25 and functional analyses. A complete ribozyme library of this design, with
12 random
positions, would contain 41z, or 1.67 x 10', different members. For the
statistical
analysis, forty individual bacterial transformants were picked and sequenced.
This
allowed an evaluation of the complexity of the library, without having to
manually
sequence each library member. The statistical "randomness" of the library was
30 determined utilizing the formula for a two-sided approximate binomial
confidence
interval: E= 1.96*squareroot(P* (1-P) /N), where P= the expected proportion of
each
nucleotide in a given position (this value for DNA bases equals 25% or
P=0.25); E=
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the desired confidence interval around P (i.e. P+/-E); and N= the required
sample
size (Callahan Associates, Inc., La Jolla, CA). To determine the proportion of
each
base within 5% (E=0.05), the required sample size is 289. Since each ribozyme
molecule contains twelve independent positions, the number of individual
ribozyme
genes that need to be sequenced out of the library equals 289 divided by 12,
or about
25 molecules.
The frequencies of the four nucleotides, with 95% confidence limits, in the
random positions were calculated to be G: 22.3 +6.1, A: 31.9+7.0, T: 27.3+7.8
and
C: 18.01+15.1. Since the expected frequency for each base is 25%, each base
appears
to be randomly represented (except for C, which may be slightly lower than
expected). These variations most likely result from the unbalanced
incorporation of
nucleotides during the chemical synthesis of the oligonucleotides and could
reduce
the complexity of the library.
For a functional evaluation of the library's complexity, in vitro cleavage was
utilized to determine if ribozymes that target known RNA substrates were
present in
the library pool. This involved in vitro transcribing of the entire ribozyme
library in
one reaction and then testing the pool's ability to cleave a variety of
different RNA
substrates of both cellular and viral origin. Six out of seven known RNA
targets were
properly and efficiently cleaved by the in vitro transcribed library. This
qualitative
analysis suggested a significantly complex library of ribozyme genes and the
lack of
cleavage of one target out of seven may reflect the slight non-randomness
suggested
by the base composition described above.
Viral vector was produced from the ribozyme library plasmid using a triple
transfection technique. CF2 cells were, seeded at 3.5 x 104 cells/cm2 one day
prior to
transfection. The cells were transfected with a l:l:lmixture of the ribozyme
library
plasmid or control ribozyme plasmid, a plasmid encoding the moloney-murine
virus
gag-pol genes, and a plasmid encoding the vesicular stomatitis virus-G gene,
using
the cationic lipid TransIT-LT1 (Pan Vera Corporation). 7.8 x 10~ cells were
transfected with 25 mg of each plasmid complexed with 250 ml of the lipid in a
total
volume of 20 ml of serum free media. After 6 hours, the media was replaced
with
growth media. The cell supernatant containing retroviral particles was
collected
every 24 hours beginning on day 2 after addition of fresh media. The
supernatant
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was filtered through 0.4 mm filters and titered in a standard assay using
HT1080
cells.
EXAMPLE II:
Isolation of ribozymes that target BRCA-1 regulator nucleic acids
This example demonstrates the isolation of ribozyme genes that bind to and
inactivate BRCA-1 regulator nucleic acid molecules, and the identification of
the
nucleic acid sequences they target.
A reporter gene based cellular selection system was used in order to
facilitate
functional selection for cells with a change in their BRCA-1 expression. The
reporter
construct allowed expression of EGFP under control of the BRCA-1 promoter
region
contained in a 2.9 kb fragment from the BRCA-1 genomic sequence. The 2.9 kb
BRCA-1 promoter region was amplified using a primer with an added PstI site
(5'-
ATCTTTCTGCAGCTGCTGGCCCGG-3'; SEQ ID NO: 52) and an AgeI
containing primer (5'-GTGTAAACCGGTAACGCGAAGAGCAGATA-3'; SEQ ID
NO: 53) and the PCR Long Template System (Boehringher Mannheim) from a
pGL2 vector containing a 3.8 kb genomic BRCA-1 fragment. The PCR product was
gel purified, digested with PstI and AgeI and cloned into pEGFP-1 (Clontech)
which
also contains a neomycin resistance marker using standard molecular
techniques.
The ovarian cancer cell line PA1 was transfected with the BRCA/EGFP
reporter construct using Lipofectamine Plus (Gibco-BRL) and stable integrants
selected using neomycin at 400-800 mg/ml for two weeks. Clones of reporter
cells
were generated by limited dilution and characterized for endogenous BRCA-1 and
EGFP expression. A reporter clone designated PBEGBR3 with an intermediate
level
of expression of both genes was chosen for library introduction. Retroviral
library
vector or control vector was introduced into the PBEGBR3 reporter cells, and
selected for stable integration of the vector using the puromycin resistance
marker. 1.
9 x 10' cells were transduced with retroviral vector (library or control) at
an MOI of
1 based on the HT1080 titer. Cells were split at a 1:2 ratio two days after
transduction and refed with media containing 0.3 mg/ml puromycin for 14 days
to
select cells with stable integration of the retroviral vector.
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After stable selection, the cell populations were subjected to several rounds
of
fluorescence activated cell sorting (FACS), enriching for cells with an
increase in
EGFP expression (the highest three to ten percent of respective populations).
After
three rounds of selection, the ribozyme library transduced population revealed
a
subpopulation with an increase in EGFP expression, while the control
transduced
cells did not. After a further round of selection, the majority of the library
transduced
cells had shifted (FIGURE 3), with a five-fold increase in mean fluorescence
intensity (MFI) compared to control vector transduced cells (FIGURE 4). RNA
analysis from these populations revealed an increase of EGFP message as well
as
endogenous BRCA-1 message in the library populations compared to control
vector
transduced cells (FIGURE S). These data underline the ability of selected
ribozymes
to increase the reporter gene expression concomitant with endogenous BRCA-1
expression, indicating a regulation on the transcriptional level.
Ribozymes were rescued from the shifted cell population by PCR
amplification. PCR rescue was performed on five separate aliquots of 1 mg of
genomic DNA extracted from the cells using the QIAmp Blood Kit (Qiagen,
Valencia, CA). PCR was carried out using the AmpliTaq Gold system (Perkin-
Elmer, Norwalk, CT) with an initial denaturation at 94°C for 10 min.
followed by 35
cycles of 94°C for 20 sec., 65°C for 30 sec., and 72°C
for 30 sec. A final extension
was performed at 72°C for 7 min. PCR primers (5'-
GGCGGGACTATGGTTGCTGACTAAT-3', 5'-
GGTTATCACGTTCGCCTCACACGC-3'; SEQ m NOS: 54 and 55, respectively)
within the vector amplified a 300 by fragment containing the ribozyme genes.
The
pooled PCR product, which contained a pool of ribozyme genes, was isolated by
electrophoresis on 1% agarose, purified using a Gel Extraction Kit (Qiagen),
then
digested with BamHI and MIuI and ligated into LHPM digested with the same
enzymes. The ligated DNA was used to transform DH12S bacteria by
electroporation. The entire bacterial culture was plated on LB-agar plates
containing
ampicillin and incubated at 37°C overnight. The resulting bacterial
colonies were
pooled and purified DNA was used in the triple transfection protocol to
generate new
retroviral vector. Individual colonies were also sequenced by the standard
dideoxy
method using a vector primer (5'-CTGACTCCATCGAGCCAGTGTAGAG-3';
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SEQ ID NO: 98). Several ribozyme sequences were found to be enriched. The
retroviral pool was introduced into new reporter cells and subjected to
several rounds
of selection by FACS. Again the library transduced cells shifted compared to
control
transduced cells. Rescue of the ribozymes from these cells showed six
predominant
ribozymes designated. RH1, RH2, RH3, RH4, RHS, and RH6 (Tables 1-6).
The substrate binding sequence of RH1 (SEQ ID NO: 5), together with its
corresponding target sequence tag (TST1; SEQ ID NO: 11) is presented in Table
1
below.
Table 1
RHl gene sequence Corresponding TST1
CCGGATGC AGAA CAAT (SEQ ID ATTG NGTC GCATCCGG (SEQ ID
NO: 5)
NO: 11)
The substrate binding sequence of RH2 (SEQ >D NO: 10), together with its
corresponding target sequence tag (TST2; SEQ >D NO: 16) is presented in Table
2
below.
Table 2
RH2 gene sequence Corresponding TST2
CCCTATTT- AGAA TTGT (SEQ ACAA NGTC AAATAGGG (SEQ ID
ID
NO: 10) NO: 16)
The substrate binding sequence of RH3 (SEQ )D NO: 6), together with its
corresponding target sequence tag (TST3; SEQ ID NO: 12) is presented in Table
3
below.
Table 3
RH3 gene sequence Corresponding TST3
AGTACATT AGAA TACT (SEQ ID, AGTA NGTC AATGTACT (SEQ ID
NO: 6) NO: 12)
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The substrate binding sequence of RH4 (SEQ ID NO: 7), together with its
corresponding target sequence tag (TST4; SEQ ID NO: 13) is presented in Table
4
below.
5 Table 4
RH4 gene sequence Corresponding TST4
CTAGTGAG AGAA GGGA (SEQ ID TCCC NGTC CTCACTAG (SEQ ID
NO: 7) NO: 13)
The substrate binding sequence of RHS (SEQ ID NO: 8), together with its
corresponding target sequence tag (TSTS; SEQ ID NO: 14) is presented in Table
5
below.
Table 5
RHS gene sequence CoiTesponding TSTS
TGAGATCC AGAA AAGC (SEQ ID GCTT NGTC GGATCTCA (SEQ ID
NO: 8) NO: 14)
The substrate binding sequence of RH6 (SEQ ID NO: 9), together with its
corresponding target sequence tag (TST6; SEQ ID NO: 15) is presented in Table
6
below.
Table 6
RH6 gene sequence Corresponding TST6
TGTTACT AGAA TGTT (SEQ ID AACA NGTC AGTAACA (SEQ ID
NO: 9) NO: 15)
To verify the function of the predominating ribozymes, vector was generated
from plasmids encoding individual ribozymes and introduced by retroviral
transduction into a second reporter system (SKHYTK4) with. the hygromycin
resistance gene driven by the BRCA-1 promoter in the SKBR3 breast carcinoma
cell
line. The BRCA-1/EGFP plasmid was used to excise a SapI insert containing the
2.9
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kb BRCA-1 promoter region, the ends were blunted using Klenow and the insert
digested with BgIII. A plasmid containing the hygromycin resistance gene fused
to
HSV thymidine kinase was used as a backbone 30 to construct the hygromycin
reporter plasmid. This plasmid contains a separate neomycin resistance
cassette. The
plasmid was digested with NheI, the ends were blunted using Klenow and the
plasmid digested with BgIII. The insert was ligated into the plasmid with one
blunt
end and one BgIII site to generate a BRCAI promoter driven hygromycin gene.
The
breast cancer cell line SKBR3 was transfected with the BRCA/HyTK reporter
construct using Lipofectamine Plus (Gibco-BRL) and stable integrants selected
using
neomycin at 400-800 mg/ml for two weeks. Clones of reporter cells were
generated
by limited dilution and characterized for resistance to hygromycin B. One
clone,
SKHYTK4, was selected for its sensitivity to hygromycin over a range of 400-
800
mg/ml. The BRCA-1/hygroTK cassette was determined to be present in these cells
by PCR and sequence analysis of the genomic DNA. SKHYTK4 cells transduced
with individual ribozymes RH3, RH4, or RH6 showed increased resistance to
hygromycin as determined by an increase in cell number when the transduced
cells
were grown in hygromycin containing media (3.1, 8.5, and 147 fold
respectively).
RH2 did not confirm in this assay.
In addition, individual ribozymes were introduced into PA1 cells by retroviral
transduction, and analyzed for their ability to grow in soft agar. 6 well
plates were
coated with Iscove's media (Gibco-BRL) containing 0.6% agar and 10% fetal calf
serum. 1 x106 PA1 cells were transduced with retroviral vector at an MOI of 1
on
day I and 2. On day 3, freshly transduced PA1 cells were overlaid on coated
plates at
6 x 104 or 9 x 104 cells per well in Iscove's media containing 0.4% agar and
7.7%
fetal calf serum. On day.4, an upper layer of Iscove's media containing 0.6%
agar
and 10% fetal calf serum was applied. Every third day, Iscove's media
containing
10% fetal calf serum was replaced on the surface of the agar layer. The cells
were
incubated for 6 weeks at 37°C, 5% C02 and the number of colonies
greater than 100
cells in size was determined. PA1 cells transduced with individual ribozymes
RH1,
4, or 5 showed a decreased ability to grow in soft agar, indicating
upregulation of the
endogenous BRCA-1 message leading to suppression of anchorage-independent
growth (3.7, 26, and 28% of control respectively). RH2 did not confirm in this
assay.
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In view of their ability to reproducibly cause an increase in expression from
the BRCA-1 tumor suppressor promoter and to induce changes in the ability of
PA1
cells to grow in soft agar, ribozymes containing substrate binding sequences
designated SEQ ID NOS: 5-10 are ribozymes which target and inactivate
regulators
of BRCA-1 expression nucleic acid molecules. Likewise, the targets of these
ribozymes, which are nucleic acid molecules containing nucleic acid sequences
designated SEQ ID NOS: 11-16, are regulators of BRCA-1 expression nucleic acid
molecules.
EXAMPLE III
Isolation and characterization of Breast Basic Conserved Protein 1 (BBC1)
This example demonstrates the isolation of a full-length transcriptional
regulator of BRCA-1 nucleic acid molecule designated Breast Basic Conserved
Protein 1 cDNA and its encoded polypeptide.
Since ribozymes recognize their cognate targets by sequence
complementarity, the sequence of a ribozyme that causes a phenotype through
its
catalytic activity predicts a sequence tag that can be used to clone the
target gene.
This "Target Sequence Tag" or TST is about 16 bases long, consisting of the
two
target regions complementary to ribozyme helices 1 and 2 and the requisite GUC
(see, for example Figures 1, 2 and 7). The TST can thus be used to BLAST
search
the gene and EST databases, and also can be used as a primer for 3' and 5'
RACE.
BLAST searches of the databases with the TST corresponding to RH1 yielded no
complete matches, and incomplete matches only with non-human sequences.
In light of the absence of obvious database candidates, the RH1 target gene
was cloned using the RK TST as a primer for a novel technique developed for
the
purpose of identifying ribozyme cleavage targets. This novel technique called
SMART C-SPACE for Switching Mechanism At 5' end of RNA Template in
Cleavage-Specific Amplification of cDNA Ends takes advantage of the unique
cleavage site of the hairpin ribozyme to generate a 5' terminus on the target
nucleic
acid consisting of the bases GUC.
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This method consists of in vitro cleavage of cellular mRNA by a particular
ribozyme (compared with a control ribozyme cleavage performed with another
aliquot of the target cell mRNA), combined with SMART cDNA technology
(Clontech) for detection of the cleavage products by PCR amplification. SMART
cDNA technology is a PCR-based method for generating high yields of high
quality,
double-stranded cDNA from nanograms of total or poly A+ RNA. Poly A+ RNA
from PA1 cells was first cleaved with in vitro transcribed RH1 ribozyme (Welch
P et
al., 1997), then used as a template for SMART cDNA synthesis. The first strand
reaction was primed by a modified oligo (dT) oligonucleotide and catalyzed by
MMLV reverse transcriptase (Superscript, GIBCO BRL). When the enzyme reaches
the 5' end of the cleaved (or uncleaved) mRNA, the enzyme's terminal
transferase
activity adds 3-5 deoxycytidine nucleotides to the 3' end of the first strand
cDNA.
The 3' end of the SMART oligonucleotide, which contains a stretch of G
residues,
anneals with the C-rich motif, forming an extended template. The reverse
transcriptase enzyme then switches templates and replicates the
oligonucleotide.
After RNase treatment, the result is a single-stranded cDNA with a sequence
complementary to the oligo (dT) primer at the 5' end and a sequence
complementary
to the SMART oligonucleotide at the 3' end. These sequences subsequently serve
as
priming sites in PCR amplification. In the case of ribozyme cleaved RNA
fragments,
the 3' attached sequence lies directly adjacent to the CAG sequence generated
by
reverse transcription of the ribozyme recognition triplet GUC of the mRNA.
This
sequence stretch, in combination with the adjacent 8 bases of helix 1 of the
ribozyme
consists of a stretch of 14 bases specific for cleaved substrate RNA
molecules. The
cDNA generated using this technique was amplified by PCR using an
oligonucleotide primer that recognize the SMART sequence, attached C residues,
GTC site, and adjacent helix 1 of the ribozyme, and an oligonucleotide primer
that
recognizes a unique portion of the oligo(dT) primer. The amplification
products were
compared between the RH1 cleaved mRNA and mRNA cleaved with a control
ribozyme. Several bands specific for the RH1 ribozyme cleaved sample were
recovered, cloned and sequenced (Figure 6).
The sequence of one of the SMART C-SPACE bands matched that of the
Breast Basic Conserved Protein 1(GenBank accession number X64707), with a
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13/16 match at the target recognition site (Scheme 2). This gene was
originally
identified by differential screening of a human breast carcinoma cDNA library.
It has
been found to be more abundant in fibroadenomas than in carcinomas. It encodes
two potential nuclear localization signals. The protein encoded by this gene
contains
a 25 amino acid region which exhibits strong similarity with the plant basic
peptide
P14, which represents a new class of transcriptional activators. It is
localized on
chromosome 16q24.3. The BBC1 message is found to be downregulated in hormone
refractory prostate cancer.
Scheme 2. Comparison of RH1 TST seduence (SEQ ID NO: 11) and BBC1
sequence (SEQ ID NO: 1)
RHI 5'-ATTG NGTC GCATCCGG-3'
BBC1 nt 208-223 5'-GTCG GGTC CCATCCGG-3'
To confirm the involvement of BBC1 in the transcriptional regulation of
BRCA-1, additional ribozyme target GUC sites were identified in the BBC1
sequence, and target validation ribozymes designed (TV6-10, Table 7; SEQ ID
NOS: 56 through 60, respectively). Target validation ribozyme expressing
retroviral
vectors generated as before from the individual target validation ribozyme
sequences
were tested as described above in the SKHYTK4 hygromycin resistance assay.
Several of these target validation ribozymes scored positive for enhanced
hygromycin resistance with TV9 showing the most pronounced effect (20 fold
increase in cell number). Additional target validation ribozymes are provided
in
Figure 8 (SEQ ID NOS: 102 through 221).
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Table 7
Validation ribozyme Gene sequence (SEQ 1D NO)
TV6 GGCTTCAA AGAA ATGC (16)
TV7 TGGGACCC AGAA CGGG (17)
TV8 CCGGATGG AGAA CGAC (18)
TV9 ACGTTCCG AGAA GGCA (19)
TV 10 CCCAGCAT AGAA GCCC (20)
To directly measure the increased expression of BRCA-1 resulting from the
5 inhibition of the transcriptional regulator BBC1, real-time PCR was
performed on
RNA from cells with and without the inhibitory ribozyme. Real-time
quantitative
polymerase chain reaction (RT-PC-R) is a process by which the cellular levels
of
mRNA encoding various proteins can be determined. In order to perform RT-PCR
three oligonucleotides based on the sequence of the mRNA of interest and a
special
10 thermal cycler with a fluorescence detector are needed. Two of these
oligonucleotides act as primers just as in regular PCR. The third
oligonucleotide acts
as a "probe" and must match sequence which lies between the two primers. This
probe has two groups attached. A reporter dye is attached to the 5' end and a
quencher is attached to the 3' end. When these two groups are close together,
as they
15 are when attached to the two ends of the oligos, the fluorescence from the
reporter
dye is quenched by the quencher. In this arrangement, the fluorescence
detector will
not be able to detect any signal. However, during PCR, the probe anneals to
its
template between the two primers. As the taq polymerase enzyme replicates the
template from the primers, it comes upon the probe. Besides its polymerase
activity,
20 taq also has a 5' to 3' exonuclease activity. Therefore, when it reaches
the place in the
template where the probe is annealed, it will displace the probe and degrade
it from
its 5' end. This releases the reporter dye from the oligonucleotide, and thus
allows the
reporter dye to diffuse away from the quencher. Once the reporter dye is
separated
from the quencher, the fluorescence detector can detect the signal. This
fluorescence
25 reading is taken at the end of each cycle of the PCR. In this way, the
detector can
determine the amount of probe which has been destroyed during each cycle. The
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amount of probe destroyed is directly proportional to the amount of template
at the
beginning of the PCR reaction. Consequently, the amount of mRNA encoding a
given protein can be determined in each sample tested. For determining BRCA-1
mRNA levels, we use the following oligonucleotide sequences:
forward primer- 5'-CTGCTCAGGGCTATCCTCTCA-3'
reverse primer- 5-TGCTGGAGCTTTATCAGGTTATGT-3
probe- 5'-1TGACATrI'TTAACCACTCAGCAGAGGGATACCA2-3'
where 1 is the 6-FAM reporter dye and 2 is the TAMRA quencher (SEQ )D NOS:
61 through 63, respectively). RT-PCR-analysis of the mRNA extracted from the
TV9
transduced cells showed a 150% increase in BRCA-1 message compared to control
vector transduced cells. This confirms the involvement of BBC1 in the observed
phenotype, and establishes a novel function of this gene as a transcriptional
regulator
of BRCA-1.
An additional SMART C-SPACE product was identified with a 12/16 match
to CHLR2 (GenBank accession number U33834), a partial sequence-found in the
database purported to be a helicase (Scheme 3). Validation of this sequence
with 5
additional ribozymes (TV 11-15, Table 8; SEQ » NOS: 65 through 69) targeting
GUC sites failed to show enhanced hygromycin resistance. This demonstrates the
requirement for the validation technology described in this application in
order to
distinguish between several possible candidate genes.
Scheme 3. Comparison of RH1 TST seauence fSEO >D NO: 11) and CHLR2
sequence (SEO >D NO: 64
RH1 5'-ATTG NGTC GCATCCGG-3'
CHLR2 pt 1621-1636 5'-GGTG GGTC GCATCCTC-3'
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Table 8
Validation ribozyme Gene sequence (SEQ ID NO)
TV 11 CCGAGAGA AGAA AGCC (65)
TV 12 TGGTTGGA AGAA CCGA (66)
TV 13 GAGGATGC AGAA CCAC (67)
TV 14 AAGAAACA AGAA ACCC (68)
TV 15 TT.GGCCAG AGAA GGGG (69)
EXAMPLE IV
Isolation and characterization of Candidate BRCA-1 Regulator (BR1)
This example demonstrates the isolation of a full-length regulator of BRCA-1
nucleic acid molecule designated Candidate BRCAI Regulator (BR1) cDNA and its
encoded polypeptide.
BLAST searches of the E8T databases with RH4 identified a 15/16 match
with an EST (GenBank accession number AA886839) without similarity to a known
gene. The RH4 TST and sequences from the identified EST were used as primers
for
5' and 3' RACE to clone a 2.9 kb cDNA (Figure 7). This novel sequence was
designated Candidate BRCA-1 Regulator (BR1). The sequence has an ORF of 127
amino acids with significant similarity to elongation factor g. However the C
terminal domain of the ORF is not conserved. This can indicate a function for
this
gene in the regulation of translation of the BRCA-1 message. Interestingly,
the PTI-
loncogene is a truncated elongation factor la.
To confirm the involvement of BR1 in the regulation of BRCA-1, additional
ribozyme target GUC sites were identified in the BR1 sequence, and target
validation
ribozymes designed (TV1-5, Table 9; SEQ ID NOS: 70 through 74, respectively).
Retroviral vectors generated as before from the individual target validation
ribozyme
sequences were tested in the SKHYTK4 hygromycin resistance assay. Several of
these target validation ribozymes scored positive for enhanced hygromycin
resistance, with TV3 showing a 17 fold increase in cell number. This confirms
the
involvement of BR1 in the observed phenotype, and establishes a novel function
of
this gene as a regulator of BRCA-1.
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Table 9
Validation ribozyme Gene sequence (SEQ ID NO)
TV 1 TTGATGTG AGAA GCTT (70)
TV2 ACTTTTCT AGAA GGAA (71)
TV3 TATTCCAT AGAA ACTG (72)
TV4 AGGACTGG AGAA AGCC (73)
TV5 AACACATT AGAA TCAA (74)
EXAMPLE V
Isolation and characterization of Inhibitor, dominant negative 4 (ID4).
This example demonstrates the isolation of a full-length transcriptional
regulator of BRCA-1 nucleic acid designated Inhibitor, dominant negative 4
(1D4)
cDNA and its encoded polypeptide.
The RH5 TST sequence showed a 14/16 match to the ID4 gene (GenBank
accession number NM_001546), by BLAST search. 1D4 is a known transcriptional
repressor located on chromosome 6p21.3-p22/6p22.3-p23. It belongs to a family
of
inhibitors of basic helix-loop-helix transcription factors. It prevents
transcriptional
activation by dimerization to the factors and blocks their ability to bind
DNA. They
are known to be involved in differentiation of adipocytes and neuronal cells.
To verify the involvement of ID4 in the regulation of BRCA-1, target
validation ribozymes (TV19-22, Table 10; SEQ ID NOS: 75 through 78,
respectively) were designed corresponding to GUC sites in the ID4 sequence.
Retroviral vectors generated from the individual target validation ribozymes
were
introduced into the SKHYTK4 cell line to assay for hygromycin resistance. The
validation ribozymes as well as the original RH5 ribozyme failed to generate
enhanced resistance to hygromycin. ID4 message was found to be undetectable in
SKBR3 cells. The validation ribozymes were tested in the soft agar assay using
PAl
cells, which do express ID4. RH5 and several target validation ribozymes
decreased
the anchorage-independent growth ability of these cells (58,10 and 34%
reduction for
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RHS, TV20, and TV21 respectively). Over-expression of the entire cDNA for ID4
in
PAl cells caused an increase in the anchorage-independent growth ability of
these
cells, leading to large colonies after 10 days of culture. RT-PCR analysis of
cells
transduced with RHS vector showed considerable variability depending on the
population tested. This can be due to a complex feedback regulation mechanism.
Table 10
Validation ribozyme Gene sequence (SEQ >D NO)
TV19 CAGTGGGC AGAA CTCA (75)
TV20 CAACAATT AGAA GGAG (76)
TV21 CACACCTG AGAA GCGC (77)
TV22 CGCGGCTG AGAA GGTC (78)
An additional possible candidate sequence was identified with a 13/16 match
to AF6 , a translocation breakpoint sequence found in AML (GenBank accession
number NM 005936). Validation of this sequence with 3 additional ribozymes
(TV23, 26, 27, Table 11; SEQ ID NOS: 79 through 81) targeting GUC sites failed
to show enhanced hygromycin resistance or growth in soft agar. This
demonstrates
the requirement for the validation technology described in this application in
order to
distinguish between several possible candidate genes.
Table 11
Validation ribozyme Gene sequence (SEQ >D NO)
TV23 GTACTAGA AGAA CGAA (79)
TV26 TGTGATCC AGAA AAGG (80)
TV27 GGTGGCCA AGAA GTGG. (81)
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EXAMPLE VI
Isolation and characterization of an EST corresponding to RH3
This example demonstrates the isolation of an EST encoding a nucleic acid
5 containing a TST corresponding to RH3.
The RH3 TST sequence was used as a primer for 5' RACE using standard
methodology. The resulting PCR product had a 13/16 match with an EST from the
databases (GenBank accession number AL045940). This EST is undergoing target
validation with 5 additional GUC recognition sites (SEQ ID NOS: 82 through 86,
10 respectively).
Table 12
Validation ribozyme Gene sequence (SEQ >D NO)
TV28 AAAAATTA AGAA GTCA (82)
TV29 GCTGTCCT AGAA TCAA (83)
TV30 TGTCAAAG AGAA CACC (84)
TV31 TGCAATGA AGAA ACTG (85)
TV32 TTACAATA AGAA ACTT (86)
A second EST was identified by database search (GenBank accession
15 number AI668913) with a 14/15 match to RH3. This EST is undergoing target
validation with 4 additional GUC recognition sites (SEQ ID NOS: 87 through 91,
respectively).
Table 13
Validation ribozyme Gene sequence (SEQ >D NO)
TV33 TCCTTCCAAGAAACGG (87)
TV34 TTGGCCCTAGAATGAG (88)
TV35 TTTTTCTAAGAAGCCC (89)
TV36 TTGTCTCAGAATGCG (90)
TV37 ATCGTCAAAGAAATCA (91)
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EXAMPLE VII
Isolation and characterization of an EST corresponding to RH6
An EST was identified in the databases with a 15/15 match to RH6 (GenBank
accession no. AI276397). Target validation ribozymes generated to 2 available
GUC
sites in this sequence (SEQ ~ NOS: 92 and 93, respectively) failed to confirm
in
either the hygromycin resistance or soft agar assays indicating that an as yet
unidentified gene is the real target responsible for the observed phenotype.
Table 14
Validation ribozyme Gene sequence (SEQ ID NO)
TV 16 CTATTTAA AGAA AATT (92)
TV 17 TATTTCTT AGAA GTTC (93)
EXAMPLE VIII
Expression of BBC1, BR1, and ID4 in human cell lines
Northern analysis of mRNA extracted from the human ovarian carcinoma cell
line PA1, and the breast carcinoma cell lines MCF7 and SKBR3 was done to
determine the expression pattern of the genes. identified as BRCA-1
regulators. Total
RNA was extracted from cell cultures grown at 80% confluency using the RNAeasy
kit (Qiagen). Fifteen micrograms of total RNA was denatured in 50% formamide,
17
mM MOPS, 2.2 M formaldehyde, for 15 min. at 70°C. Samples were
separated by
electrophoresis in a 0.8% agarose gel containing 2.2 M formaldehyde and 20 mM
MOPS and transferred to a nylon membrane for hybridization with 32P labeled
probes generated from plasmids encoding either BBC1, BR1, or 1174. BBCI and
BR2 were found to be expressed in both PA1 and SKBR3 cells (MCF7 not done).
1D4 was found to be expressed in both PA1 and MCF7 but not SKBR3 cells. To
confirm this result, RT-PCR analysis using total RNA was performed as this is
a
more sensitive technique. Again, m4 was found. to be expressed in both PA1 and
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MCF7 but not SKBR3 cells. This explains the lack of effect of the TD4 targeted
ribozymes observed in the SKBR3 based hygromycin resistance assay.
EXAMPLE IX
Effect of over-expression of ID4
To validate the biological relevance of ID4 regulation of BRCA-1, ID4 sense
and antisense expression vectors were introduced into P8R3 cells by
transduction
with retroviral vectors. The cDNA sequence from ID4 was amplified using
primers
>D4-3 (5'-TCCGAAGGGAGTGACTAGGACACCC-3'; SEQ ID NO: 94) and
ID4-7 (5'-TTCTGCTCTTCCCCCTCCCTCTCTA-3'; SEQ ID NO: 95). The
amplified product was cloned into T/A-vector (Invitrogen), and verified by
sequencing from both vector sites. Plasmid DNA was digested with Eco RI,
releasing
the whole insert out of the cloning vector and cloned into the EcoRI
linearized
retroviral expression vector pLPCX (Clontech). Clones were analyzed for the
correct
insert as well as for orientation screening by multiple restriction digests.
Sequencing
of both junction sites from both directions using vector-based primers 5 - and
3 -
LPCX (Clontech) confirmed the correct orientation and junction sequence.
Following stable integration, cells were evaluated for their anchorage-
independent
growth potential using the soft agar assay described above. The biological
consequences of BRCA-1 downregulation should be increased growth in soft agar,
and the converse for BRCA-1 upregulation. Cells expressing sense 1174 had a
dramatic increase in anchorage-independent growth in comparison to cells
expressing antisense ID4.
EXAMPLE X
Detection of genes in the Regulation Pathway of BRCA-1
This example provides methods to use BRCA-1 regulator ribozymes to
identify other genes in the BRCA-1 pathway that contribute to the phenotype
changes observed after ribozyme mediated target RNA cleavage (i. e. target
"knockdown"). Expression profiling of genes in hybridization assays identifies
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cellular genes upregulated or downregulated subsequent to BRCA-1 regulator
knockdown. Such genes constitute potential BRCA-1 regulators.
RNA extracted from cells expressing ribozymes RH1, RH3, RH4, RHS, and
RH6 was sequentially hybridized to an array filter. Probes, filters, and
analysis were
as recommended by the manufacturer (Research Genetics, Huntsville AL).
Briefly,
micrograms of total RNA from each ribozyme or control transduced cell
population was primed with oligo dT, and reverse transcribed with reverse
transcriptase (Superscript II Life Technologies, Gaithersburg MD)
incorporating 33P
labeled dCTP to generate a detectable cDNA probe. Denatured probe was
10 hybridized to the filter in the supplied buffer for 18 hours at
42°C. Filters were
washed in 2X SSC, 1°lo SDS at 50°C twice for 20 minutes each,
followed by washing
in 0.5 X SSC, 1°7o SDS at room temperature for 15 minutes. The filters
were
exposed to a phospor imaging screen and the image captured using a Molecular
Dynamics (Sunnyvale, CA) phosphor imaging system. Comparisons between filters
were made using the Pathways analysis software (Research Genetics, Huntsville
AL).
The expression profile was compared to the pattern generated by hybridizing
the filter to RNA extracted from cells expressing the control ribozyme.
Several
genes were found to be down-regulated in some or all of the ribozyme
expression
profiles. From the pattern of expression it is possible to ascertain a pathway
of
differentially expressed genes. Numerous genes were differentially expressed
at
levels in the ribozyme expression cells that were greater than in the control
cells.
1. AA419229: EST sharing sequence identity with orphan GPR39
A. AF034633.1 - Homo sapiens orphan G protein-coupled receptor (GPR39)
mRNA, complete cds
B. 4504096 - Homo sapiens G protein-coupled receptor 39 (GPR39) mRNA,
and translated products
C. Protein: 043194 - Human putative G protein-coupled receptor 39
D. Protein: 4504097 - G protein-coupled receptor 39
2. H18950: EST sharing sequence identity with NADC3
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A. AF154121.1 - Homo sapiens sodium-dependent high-affinity dicarboxylate
transporter (NADC3) mRNA, complete cds
3. H07920: EST sharing sequence identity with MKK6
A. ~ 012648.1 - Homo sapiens mitogen-activated protein kinase kinase 6
(MAP2K6), mRNA
B. NM-002758.1 - Homo sapiens mitogen-activated protein kinase kinase 6
(MAP2K6), mRNA
C. U39657.1 - Human MAP kinase kinase 6 (MKK6) mRNA, complete cds
4. H70047: EST sharing sequence identity with RGS 13
A. AF030107 - Homo sapiens regulator of G protein signaling (RGS 13) mRNA,
complete cds
5. H84815: EST sharing sequence identity with Rab9 effector p40
A. BC000503 - Homo sapiens, Rab9 effector p40, clone MGC:8459, mRNA,
complete cds
B. NM-005833.1 - Homo sapiens Rab9 effector p40 (RAB9P40), mRNA
C. 297074.1 - Homo sapiens mRNA for Rab9 effector p40, complete cds
6. AA757764: EST sharing sequence identity with multi Zn-finger TF
A. D49835 - Homo Sapiens mRNA for DNA-binding protein, complete cds
7. H19111: EST sharing sequence identity with sialyltransferase
8. AA629897: EST sharing sequence identity with laminin receptor
A. M14199.1 - Human laminin receptor (2H5 epitope) mRNA, 5' end
B. BC002533 - Homo sapiens, laminin receptor 1 (67kD, ribosomal protein SA),
clone MGC:2208, mRNA, complete cds
C. NM 002295.2 - Homo sapiens laminin receptor 1 (67kD, ribosomal protein
SA) (LAMR1), mRNA
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9. AA663439: EST sharing sequence identity with adenine nucleotide
translocator 3
A. J03592.1 - Human ADP/ATP translocase mRNA, 3' end, clone pHAT8
B. AF076617 - Homo sapiens ADP/ATP translocase mRNA, 3'UTR
C. L29034.1 - Human mitochondria) (clone GC6-T5) ADP/ATP translocase
5 gene sequence
10. 815740: EST sharing sequence identity with chondroitin-6-sulfotransferase
A. XM_012024.1 - Homo sapiens carbohydrate (chondroitin 6/keratan)
sulfotransferase 1 (CHST1), mRNA
10 B. NM_003654.1 - Homo sapiens carbohydrate (chondroitin 6/keratan)
sulfotransferase 1 (CHST1), mRNA
C. U65637.1 - Homo sapiens chondroitin-6-sulfotransferase mRNA, complete
cds
15 11. AA454570: EST sharing sequence identity with lambda 5 semaphorin
A. U38276.1 - Human semaphorin III family homolog mRNA, complete cds
12. AA485748: EST sharing sequence identity with fibromodulin
A. XM_001782.2 - Homo sapiens fibromodulin (FMOD), mRNA
20 B. NM_002023.2 - Homo Sapiens fibromodulin (FMOD), mRNA
13. AA464601: EST sharing sequence identity with tspan-5
A. AF065389 - Homo sapiens tetraspan NET-4 mRNA, complete cds
B. AF053455 - Homo sapiens tetraspan TM4SF (TSPAN-5) gene, complete cds
25 C. NM_005723.1 - Homo sapiens tetraspan 5 (TSPAN-5), mRNA
D. XM_011178.1 - Homo sapiens sharing sequence identity with tetraspan 5 (H.
Sapiens) (LOC65435), mRNA
14. AA102107: EST sharing sequence identity with aminopeptidase A
30 A. L12468.1 - Homo Sapiens aminopeptidase A mRNA, complete cds
15. 249826: HSHNF4G hepatocyte nuclear factor 4 gamma
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Ribozymes can be designed to target the cleavage sites in the above 15 genes
to verify that the above genes are BRCA-1 regulators according to methods
disclosed
above. Potential cleavage TST target sequences and the sequence of a ribozyme
substrate recognition site for cleaving those TST target sequences is shown in
tables
15-17 below.
Table 15: Validation RST and TST sequences for GenBank Acc. No. H07920
Validation ribozyme RST TST target sequence
3'-AAGG UCAG ACAAAACG'-5' 5'-TTCC AGTC TGTTTTGC-3'
(SEQ >D NO: ) (SEQ ID NO: )
3'-GUAG CCAG UUCUCUUU-5' S'-CATC GGTC AAGAGAAA-3'
(SEQ >D NO: ) (SEQ m NO: )
3'- CUUG ACAG AUCUACCU-5' S'-GAAC TGTC TAGATGGA-3'
(SEQ >I7 NO: ) (SEQ ID NO: )
Table 16: Validation RST and TST sequences for GenBank Acc. No. AA419229
Validation ribozyme RST TST target sequence
5'-AGUG UCAG UACAGGGG-3' S'- TCAC AGTC ATGTCCCC -3'
(SEQ >D NO: ) (SEQ ID NO: )
3 '-GGGG UCAG AUUCAGGG-5' S'- CCCC AGTC TAAGTCCC -3'
(SEQ m NO: ) (SEQ m NO: )
3'- UAAC UCAG AGCUCAGU-5' S'- ATTG AGTC TCGAGTCA -3'
(SEQ ID NO: ) (SEQ ID NO: )
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Table 17: Validation RST and TST sequences for GenBank Acc. No. 249826
Validation ribozyme RST TST target sequence
5'-UCGG UCAG ACUCUAUA-3' (SEQ5'- AGCC AGTC TGAGATAT -3'
>Z.7 NO: ) (SEQ ID NO: )
3 '-UGCC ACAG UUGACAGA-5' S'- ACGG TGTC AACTGTCT-3'
(SEQ >D NO: ) (SEQ >D NO: )
3'- GGUC CCAG UUCGUGAC-5' S'- CCTG GGTC AAGCACTG -3'
(SEQ ID NO: ) (SEQ ID NO: )
3'- ACCG UCAG UAGAGGUA-5' S'- TGGC AGTC ATCTCCAT -3'
(SEQ ID NO: ) (SEQ ID NO: )
3'- GUAG UCAG UAAAGUGU-5' S'- CATC AGTC ATTTCACA -3'
(SEQ ID NO: ) (SEQ ID NO: )
***********
Throughout this application various publications have been referenced within
parentheses. The disclosures of these publications in their entireties are
hereby
incorporated by reference in this application in order to more fully describe
the state
of the art to which this invention pertains.
Although the invention has been described with reference to the disclosed
embodiments, those skilled in the art will readily appreciate that the
specific
experiments detailed are only illustrative of the invention. It should be
understood
that various modifications can be made without departing from the spirit of
the
invention. Accordingly, the invention is limited only by the following claims.
