Note: Descriptions are shown in the official language in which they were submitted.
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METHODS FOR IDENTIFYING THE TOXIC/PATHOLOGIC EFFECT
OF ENVIRONMENTAL STIMULI ON GENE TRANSCRIPTION
The present invention relates to the use of arrays or grids of mammalian gene
sequence fragments from genomic (or cDNA) libraries for the screening of
environmental factors, such as pharmaceutical compounds, physical factors,
infectious agents, etc, for a toxic or pathologic effect upon gene
transcription.
Mammalian cells frequently respond to exogenous stimuli of many types by
altering the rate of transcription. For example, exposure of mammalian cells
to
environmental factors such as ultraviolet light, pharmaceutical compounds and
many
others can increase or decrease the quantity of messenger RNA produced by the
cells. These changes in transcriptional regulation can result in toxic or
pathological
responses by the mammal. For example, where the external stimuli is prolonged
exposure to UV rays, the toxic response of the mammal can be sunburn. Where
the
external stimuli is a compound known to be hepatotoxic, the response is liver
damage. Where the external stimuli is a carcinogen, the toxic response is
uncontrolled growth of cells.
The development of new pharmaceutical compositions and/or treatment
regimens directed towards the treatment or prophylaxis of a variety of
diseases,
infectious or otherwise, relies quite heavily on the ability to screen
candidate
reagents for possible toxic or pathologic response. In normal drug development
a
novel chemical compound, novel biological composition, and the like is run
through
a battery of assays in vitro and in laboratory animals to ascertain its safety
(i.e., lack
of toxicity) and effectiveness.
The costs associated with the development of new pharmaceutical reagents
are ever increasing, particularly when new compositions enter clinical trials.
It is not
unknown for promising pharmaceutical candidates to pass the appropriate
laboratory
tests and enter the expensive stage of animal and human clinical trials, only
to
present toxic or pathologic effects in the in vivo setting for the targeted
mammalian
patient, normally humans. The elimination of previously promising drug
candidates
at such a late stage in product development is a major factor in the high
costs of new
effective drugs which ultimately do pass the final clinical trials. Such late
elimination of toxic compounds also results in unnecessary human suffering and
wasted effort.
Methods have been described for obtaining information about gene
expression and identity using so called "high density DNA arrays" or grids.
See,
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e.g., M. Chee et al, Science, 274:610-614 (1996) and other references cited
therein.
Such gridding assays have been employed to identify certain novel gene
sequences,
referred to as Expressed Sequence Tags (EST) [Adams et al., Science, _252:1651-
1656 (1991)]. A variety of techniques have also been described for identifying
particular gene sequences on the basis of their gene products. For example,
see
International Patent Application No. W091/07087, published May 30, 1991. In
addition, methods have been described for the amplification of desired
sequences.
For example, see International Patent Application No. W091/17271, published
November 14, 1991.
Accordingly, there exists a need for more efficient methods for screening
novel pharmaceutical reagents, as well as other environmental stimuli or
factors, to
identify any toxic/pathogenic effect on gene transcription for both new drug
development and new therapeutic regimens.
In one aspect, the invention provides a method of assessing the genetic effect
of a selected environmental factor on a mammalian subject, said method
comprising
the steps of:
(a) providing a plurality of identical grids, each grid comprising a surface
on which is immobilized at predefined regions on said surface a plurality of
unique
defined gene sequence fragments, said oligonucleotide sequences comprising
genes
or fragments of genes obtained from a healthy member of said mammalian
species;
(b) exposing mammalian cells, tissue or organ to an environmental factor
for a sufficient time to affect transcription of messenger RNA in said cells;
(c) extracting and isolating mRNA from said exposed cells, tissue or
organ of step (b);
(d) extracting and isolating control mRNA from mammalian cells, tissue
or organ not exposed to said factor;
(e) labelling the mRNA from steps (c) and (d);
(f) hybridizing the labeled mRNA from the exposed cells, tissue or organ
to a first identical grid to produce a first hybridization pattern detectable
by an
increased quantity of fluorescence in contrast to the remainder of the grid;
(g) hybridizing the labeled control mRNA to a second identical grid to
produce a second, control hybridization pattern; and
(h) comparing the first and second hybridization patterns to identify any
change in said first pattern from the control pattern, indicative of an effect
on
3 ~ transcriptional regulation of said mammalian cells, tissue or organ
exposed to said
factor.
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The method of the invention thus employs the following steps. A plurality of
identical DNA grids is prepared. At predefined regions on the grid surface, a
plurality of defined amplified gene sequences (or oligonucleotide sequences)
is
immobilized. These gene sequences preferably are known or unknown genes, or
fragments of genes, obtained from the cells (or a library of cells) of a
healthy
member of the mammalian species. Messenger RNA is isolated and extracted from
mammalian cells which are not exposed to a selected environmental stimulus,
thus
forming the "control" RNA. The "test mRNA" is extracted from mammalian cells
which have been exposed for a sufficient time to affect gene transcription to
the
selected stimulus. The control and test mRNA are randomly labeled, and each
mRNA preparation is applied to an identical grid. The respective hybridization
patterns are compared to identify any change in the test pattern from the
control
pattern, indicative of an effect on transcriptional regulation of the
mammalian cells
exposed to the stimulus. The determination of stimuli having a toxic or
pathologic
effect is useful, e.g., in the screening and development of new pharmaceutical
agents
and therapies.
The arrays or grids of mammalian gene sequence fragments from genomic
(or cDNA) libraries used in the method of the invention may be high density
DNA
arrays or grids.
In another aspect, the method described above is performed for a "class" of
stimuli, e.g., chemical or pharmaceutical compounds, which are to generate a
common toxic or pathologic effect upon exposure to mammalian cells, e.g.,
hepatotoxicity. The method generates a "fingerprint" hybridization pattern for
e.g.,
hepatotoxic, stimuli. Thus, test candidate drugs compositions may be screened
for
the likelihood of causing hepatotoxicity in mammalian cells by comparing the
test
hybridization pattern to the fingerprint at an early stage in drug
development.
Similarity between the fingerprint and the test pattern permit early
elimination of the
candidate drug from consideration, thus permitting only non-hepatotoxic
compounds
to proceed to drug development.
In still another aspect, the methods of the present invention may be
performed to identify those genes which are the most responsive to a
particular toxic
effect of an external stimuli.
In still further aspects, the invention provides methods of identifying
possible
toxic or pathological effects of a variety of disparate physical stimuli, as
well as
3~ chemical and pharmaceutical stimuli.
Other objects, features, advantages and aspects of the present invention will
become apparent to those of skill in the art from the following description.
It should
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be understood, however, that the following description, while indicating
preferred
embodiments of the invention, are given by way of illustration only. Various
changes and modifications within the spirit and scope of the disclosed
invention will
become readily apparent to those skilled in the art from reading the following
description and from reading the other parts of the present disclosure.
The present invention meets the needs of the art by providing a method of
assessing the effect of any environmental factor or stimulus on gene
expression in a
mammalian subject by using DNA gridding techniques. Such techniques, employed
as described below, permit the identification of genes which display a
response to a
test compound, permit the identification of a hybridization pattern
characteristic of
known physiologic effect in response to a test compound and permit the
"fingerprinting" of certain selected toxic effects. The fingerprints are
useful in
screening new compounds or drug candidates for potential toxicity and in
screening
for the effect on gene transcription of other environmental stimuli. The
information
generated thereby can be used in the phanmaceutical industry to identify new
drugs,
in occupational safety evaluations of the workplace environment, and in many
other
industries and settings where it may be necessary to take measures to correct
environmental stimuli which cause adverse effects in humans, and other
mammals.
Several words and phrases used throughout this specification are defined as
follows:
As used herein, the term "gene" refers to the genomic nucleotide sequence
from which a cDNA sequence is derived. The term gene classically refers to the
genomic sequence, which upon processing, can produce different RNAs.
By "gene product" it is meant any polypeptide sequence, peptide or protein,
encoded by a gene. The term "genomic library" is meant to include, but is not
limited to, plasmid libraries, PCR products from genomic libraries, cDNA
libraries
and known sequences. Methods for the construction of such libraries are well
known by those skilled in the art. A genomic library may be adjusted to
minimize
the number of complete genes present in a single genomic insert to
approximately
one gene. Techniques for this adjustment are well known to the skilled
artisan.
"Isolated" means altered "by the hand of man" from its natural state; i.e.,
that,
if it occurs in nature, it has been changed or removed from its original
environment,
or both. For example, a polynucleotide or a polypeptide naturally present in a
living
animal in its natural state is not "isolated," but the same polynucleotide or
polypeptide separated from the coexisting materials of its natural state is
"isolated,"
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as the term is employed herein. For example, with respect to polynucleotides,
the
term isolated means that it is separated from the chromosome and cell in which
it
naturally occurs.
"Pathogenic effect" or "pathologic effect", as used herein, refers to a change
in gene expression which may cause a disease or disorder. The change is due to
exposure of a mammal or mammalian cell to some environmental stimulus, as
detailed below.
As used herein, the term "solid support" refers to any substrate which is
useful for the immobilization of a plurality of defined materials derived from
a
genomic library by any available method to enable detectable hybridization of
the
immobilized polynucleotide sequences with other polynucleotides in the sample.
Among a number of available solid supports, one desirable example is the
support
described in International Patent Application No. W091/07087, published May
30,
1991. Examples of other useful supports include, but are not limited to,
nitrocellulose, nylon, glass, silica and Pall BIODYNE C membrane. It is also
anticipated that improvements yet to be made to conventional solid supports
may
also be employed in this invention.
The term "grid" means any generally two-dimensional structure on a solid
support to which the defined materials of a genomic library are attached or
immobilized. Preferably according to this invention, three types of grids are
useful.
One grid useful in this invention contains as its defined oligonucleotide
materials,
unique nucleic acid sequences (or "tags"; or expressed sequence tags ("EST")]
from
all human genes identified. A second useful grid contains unique nucleic acid
ESTs
from genes cloned from a tissue or a cell line. Still a third type of grid
useful in the
present invention contains unique nucleic acid tags from genes classified as
particularly relevant to identification of a selected environmental toxicity.
Grids are
desirably constructed from animal species used in the preclinical assessment
of
compound safety.
As used herein, the term "predefined region" refers to a localized area on a
surface of a solid support on which is immobilized one or multiple copies of a
particular amplified gene region or sequence and which enables hybridization
of that
clone at the position, if hybridization of that clone to a sample
polynucleotide
occurs.
By "immobilized," it is meant to refer to the attachment of the genes to the
solid support. Means of immobilization are known and conventional to those of
skill in the art, and may depend on the type of support being used.
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The terms "environmental factor" or "environmental stimuli" are used herein
to describe a wide variety of physical, chemical or biological factors which
cause
changes in gene transcription in a mammalian cell when the mammal itself, or a
culture of such mammalian cells, is exposed to that factor. For example,
physical
environmental stimuli can include, without limitation, the diet of the mammal,
an
increase or decrease in temperature; an increase or decrease in exposure to
ionizing
or ultraviolet radiation, and the like. A biological/chemical stimuli can
include,
without limitation, administering a transgene to the mammal, or eliminating a
gene
from the mammal; administering an exogenous synthetic compound or exogenous
agent or an endogenous compound, agent or analog thereof to the mammal.
As an example, an exogenous synthetic compound can be a pharmaceutical
compound, a toxic compound, a protein, a peptide, a chemical composition,
among
other. An exogenous agent can include natural pathogens, such as microbial
agents,
which can alter gene transcription. Examples of pathogens include bacteria,
viruses,
and lower eukaryotic cells such as fungi, yeast, molds and simple
multicellular
organisms, which are capable of infecting a mammal and replicating its nucleic
acid
sequences in the cells or tissue of that mammal. Such a pathogen is generally
associated with a disease condition in the infected mammal.
An endogenous compound is a compound which occurs naturally in the
body. Examples include hormones, enzymes, receptors, ligands, and the like. An
analogue is an endogenous compound which is preferably produced by recombinant
techniques and which differs from said naturally occurring endogenous compound
in
some way.
By "transcriptional effect" is meant an increase or decrease in rate of
transcription in the mammalian cells exposed to the stimuli.
A "fingerprint" as used herein is defined as a characteristic hybridization
pattern on a grid indicating a common toxicological response, i.e., similar
increases
in gene transcription that result in similar tissue damage. For example, using
the
methods described herein, one may generate a "hepatotoxic" fingerprint, which
can
be used to identify compounds which are likely to have a toxic effect on the
liver,
and so on.
By "label" as used herein is meant any conventional molecule which can be
readily attached to mRNA and which can produce a detectable signal, the
intensity
of which indicates the relative amount of hybridization of the ml2NA to the
DNA
fragment (oligonucleotide) on the grid. Prefers ed labels are fluorescent
molecules or
radioactive molecules. A variety of well-known labels can be used.
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Method of the Invention:
A. The Grids
According to the present invention, a method is provided which enables the
association of selected environmental stimuli with changes in gene
transcription.
One of the specific applications of this technology is the understanding and
prediction of toxic reactions to environmental manipulations and
modifications, such
as those stimuli listed above. Another application is in pre-clinical and
clinical drug
development, where the method of this invention enables the screening of
compounds having a similar toxic effect on gene transcription by comparison to
the
effect of another stimulus.
In the practice of this method, a plurality of identical grids is prepared, so
that each grid carries on its solid surface a plurality of defined unique gene
(oligonucleotide) sequences immobilized at predef ned regions on the surface.
The
gene sequences immobilized on the grids are as defined above, i.e., as unique
nucleic
acid tags from all human or other mammalian genes, or from only a selected
tissue,
e.g., reticulocytes, or the liver, or a selected cell line, or from genes
known to be
relevant to environmental toxicity, e.g., the lung, kidney, heart, blood
cells, etc.
These genes or fragments of genes immobilized on the grids may be obtained
from
an oligonucleotide library of a healthy member of the mammalian species, e.g.,
a
healthy human. Other mammals of interest include, without limitation, a non-
human
primate, a rodent, and a canine.
For the purposes of this invention, it is not necessary that the grids reflect
a
single target organ, although such a specific target grid can be used. It is
anticipated
that the response of the mammalian cell to various environmental stimuli that
effect
gene transcription is likely to be stereotypic of genes in other cells. Thus,
the grid
can be prepared from red or white blood cells, reticulocytes, or
undifferentiated
cells, even where the particular toxicological effect is damage to the liver
or some
other particular tissue. Alternatively, such a grid can be prepared from
hepatocytes
only, or from cells from the effected organ or tissue only. All grids are
anticipated
to reflect the same hybridization pattern upon exposure to a reagent or
stimulus that
is known as hepatotoxic. The same is true regardless of the type of
toxicological
damage, e.g., cardiac damage, kidney damage, hematopoietic cell damage, etc.
The gene fragments immobilized on the grid may be obtained from a random
cDNA library of the target mammal using known techniques. Alternatively, a
3~ cDNA library of genes from a selected organ or tissue may be prepared as
the source
of the sequences immobilized on the grid. The RNA is isolated and reverse
transcribed to cDNA using standard procedures for molecular biology such as
those
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disclosed by Sambrook et al., MOLECULAR CLONING, A LABORATORY
MANUAL, 2nd Ed; Cold Spring Harbor Laboratory Press, Cold Spring Harbor Lab
Press, Cold Spring Harbor, NY 1989. The cDNA library is then constructed in
accordance with procedures described by Fleischmann et al. Science, 1995,
269:496-
S 12. For the purposes of the present invention, a cDNA library can comprise a
plasmid library, PCR products from a cDNA library, or known sequences.
A plurality of genes or gene fragments, whether known or random and
unknown, from the selected library are gridded onto a surface of a solid
support at
predefined locations or regions, preferably at 6X coverage. By "plurality of
materials derived from the genomic library" it is meant to include, but is not
limited
to, individual clones spotted onto and grown on a surface of the solid support
at
predefined locations or regions; or plasmid clones isolated from said library,
PCR
products derived from the plasmid clones, or oligonucleotides derived from
sequencing of the plasmid clones, which are immobilized to the surface of the
solid
support at predefined locations or regions. As selection of genes involved in
e.g.,
carcinogenicity, apoptosis, inflammation, metabolism of compounds etc, may be
used.
The grids used in the invention may contain, e.g., up to 5,000 genes or gene
fragments. The grids preferably contain up to 1,500 genes or gene fragments,
e.g.,
100 to 1,500 genes or gene fragments, more preferably about 1,000 genes or
gene
fragments.
Numerous conventional methods are employed for immobilizing these gene
sequences (oligonucleotides) to surfaces of a variety of solid supports. See,
e.g.,
Affinity Techniques, Enzyme Purification: Part P, Methods in Enzymology, Vol.
34, ed. W.B. Jakoby, M. Wilcheck, Acad. Press, NY (1971); Immobilized
Biochemicals and Affinity Chromatography, Advances in Experimental Medicine
and Biology, Vol. 42, ed. R. Dunlap, Plenum Press, NY (1974); U.S. Patent
4,762,881; U.S. Patent No. 4,542,102; European Patent Publication No. 391,608
{October 10, 1990); or U.S. Patent No. 4,992,127 (November 21, 1989).
One desirable method for attaching these materials to a solid support is
described in International Application No. PCT/LJS90/06607 (published May 30,
1991). Briefly, this method involves forming predefined regions on a surface
of a
solid support, where the predefined regions are capable of immobilizing the
materials. The method makes use of binding substrates attached to the surface
3~ which enable selective activation of the predefined regions. Upon
activation, these
binding substances become capable of binding and immobilizing the materials
derived from the genomic library.
_g_
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Any of the known solid substrates suitable for binding nucleotide sequences
at predefined regions on the surface thereof for hybridization and methods for
attaching nucleotide sequences thereto may be employed by one of skill in the
art
according to the invention.
As described above the genes or gene fragments may be of known or
unknown function. In a fingerprinting method it is not necessary to know the
function of every gene since the method may not be looking at specific
pathways of
toxicity but at distinct patterns of gene expression in response to
environmental
factors.
t0 B. Obtaining the mRNA for hybridization to the grids
The selected mammalian cells, tissues or organs to be examined for
transcription changes are subjected to the environmental stimulus for a
sufficient
time to affect transcription of messenger RNA in the cells. This "exposing"
step can
occur by treating or exposing a living healthy animal or human to the
stimulus. For
example, the selected mammal may be administered a reagent, such as an
exogenous
or endogenous compounds as described above. Alternatively, the mammal may be
exposed to a physical stimulus, e.g., W radiation:
Alternatively, a mammalian cell culture or tissue culture, or viable organ,
e.g., liver, heart, etc., may be exposed to the stimulus in vitro. A control
mRNA
source is an untreated animal, tissue, organ or cell culture.
The exposure to the environmental stimulus, which may be stimuli known to
cause a specific physical effect, e.g., hepatocyte damage, cancer, etc.,
occurs for a
time sufficient to result in the alteration from the normal of the
transcription level of
the cells so exposed. The sufficient time will depend upon the particular
stimulus
being studied and, in fact, determination of a sufficient stimulus time is
well within
the skill of the art.
Where the mRNA source is a cell culture, the culture is then incubated under
a selected set of defined in vitro or in vivo conditions to produce a test
culture. In
addition, non-exposed cells are also cultured under the same set of defined
conditions to produce a control culture. By "defined conditions" it is meant,
but is
not limited to, standard in vitro culture conditions recognized as normal
(i.e., non-
pathogenic) for a selected mammalian cell, as well as in vitro conditions
which
reflect or mimic in vivo pathogenic settings (conditions) such as heat shock,
auxotrophic, osmotic shock, antibiotic or drug selection/addition varied
carbon
sources, and aerobic or anaerobic conditions, and in vivo, pathogenic
conditions.
Preferably, such conditions are predetermined to allow maximum growth of the
non-
exposed cells.
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The cells are then harvested from the animal, organ, tissue or cell culture by
conventional means. Harvesting can be performed during various growth stages
of
the cells to ascertain the essentiality of a particular gene during different
stages of
growth. For example, harvesting can be performed during early logarithmic
growth,
late logarithmic growth, stationary phase growth or late stationary growth.
RNA (or
DNA) is then extracted and isolated from the harvested non-exposed cells of
the
control culture, and RNA is extracted and isolated from the cells exposed to
the
stimulus of the test culture using standard methodologies well known to those
skilled in the art.
I O mRNA extracted from the cells of the control culture and from the cells of
the test culture are then used to generate labeled probes. When mRNA from the
control and test cells is used to generate the probes, isolated mRNA is
labeled
according to standard methods using random primers, preferably hexamers, and
reverse transcriptase. Such methods are routinely performed by those skilled
in the
art. All mRNA from the "control" or the "exposed" source is randomly labeled
by
conventional means, such as nick translation, multiprime labelling or other
commonly used enzymatic labeling methodology. Known conventional methods for
labelling the mRNA sequences may be used and make hybridization of the
immobilized materials detectable. For example, fluorescence, radioactivity,
photoactivation, biotinylation, energy transfer, solid state circuitry, and
the like may
be used in this invention.
C. Hybridization to the grids
These labeled mRNAs are then used as hybridization probes against the
identical high density grids. Labeled probes prepared from mRNA extracted from
the test culture are hybridized to one grid to produce a "test" hybridization
pattern.
Labeled probes from the mRNA extracted from the cells of the control culture
are
hybridized to a second identical grid, resulting in a "control" hybridization
pattern.
The generated test hybridization patterns and control hybridization patterns
are then compared. In the control pattern, the mRNA binds to certain genes or
gene
fragments in the grid in proportion to the expression of the mRNA of such
genes in a
normal cell. The pattern is detectable by an increased quantity of detectable
signal,
e.g., fluorescence, at locations on the grid of those genes which are normally
expressed in greater quantities that others in the remainder of the grid.
In the test grid, genes for which transcription is enhanced by the stimulus
will be bound by a greater amount of labeled mRNA, and genes for which
transcription is reduced by the stimulus wilt be bound by a lesser amount of
labeled
mRNA, thus altering the hybridization pattern from that of the control.
Comparison
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of the test and control patterns reveals the effect of the test compound on
transcription of certain genes located at the predefined locations on other
grid.
D. The Fingerprints
Thus, where the test compound or stimulus is a stimulus known to cause a
physiological effect, for example, a toxic reaction of a subject resulting in
damage to
a major organ, e.g., liver, kidney, heart, blood cells, the method of this
invention
may be performed to provide a hybridization pattern which correlates with that
damage. Most desirably, for preclinical drug screening according to this
invention,
any collection of known and structurally distinct toxicants which have the
same
physiological effects, e.g., hepatotoxicity, can be employed in this method to
generate a characteristic "fingerprint" hybridization pattern for hepatotoxic
stimuli.
Where it is desired to produce a common hybridization pattern such known
toxicants, a set of grids are calibrated with a repertoire of the structurally
diverse
toxicants that produce the same pathological/toxicological reaction; e.g.
hepatotoxicity or nephrotoxicity. In other words, labeled RNA from a mammalian
cell source exposed to the known toxicants are hybridized to identical grids
to
produce a common toxicant hybridization pattern. If the variety of known
toxicants
produce a characteristic common hybridization pattern, the common
toxicological
responses are likely to be the result of similar increases in transcription of
selected
genes, resulting in similar tissue damage. This toxicological fingerprint
pattern may
be used along with the "control" pattern for comparison with the pattern of a
test
compound/stimulus of unknown function or result. Thus the common fingerprint
for, e.g., hepatotoxicity, is used to screen a stimulus of unknown function or
effect to
determine if that stimulus is likely to produce hepatotoxicity in the mammal.
Similarity in the "test" pattern to the hepatotoxic fingerprint enables the
putative identification of the test compound as a hepatotoxic compound. Thus,
if the
test compound was a drug candidate, it can be eliminated from consideration at
the
earliest stages of drug development on the basis of its effects on gene
transcription
as measured on the grids. Similarly the method permits the test compound or
stimulus, if an environmental factor present in e.g., the workplace, such as
radiation,
etc., to be identified as a potential health hazard, and corrected.
According to this method, therefore, a battery of fingerprint hybridization
patterns may be prepared for all known toxicants. Any new drug candidate or
other
environmental stimulus may be screened by the above method for probable
3~ toxicological effects by comparison to standard fingerprints for other
known stimuli
causing liver damage, kidney damage, damage to the hematopoietic systems, etc.
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Such a screening method will enable quick and early evaluation of
environmental
stimuli, particularly new drug candidates.
Fingerprint hybridization patterns may be stored in a database and pattern
matching performed by datamining.
E. Preclinical Embodiments of the Method
In a particularly desirably embodiment of the method of this invention, in
vitro effects of pharmacologically relevant concentrations of compounds on
gene
expression in blood cells are examined using the methods of this invention. A
gene
expression fingerprint ~is developed through this methodology by exposing the
nucleated blood cells, e.g., reticulocytes, white cells, to a variety of
toxicants as
described above. The resulting fingerprint is used subsequently to predict
whether a
novel compound is likely to also produce a similar pathological reaction. The
information assists decisions about which compounds to take forward to
clinical
development, and enhances safety in the clinic through accurate and early
prediction
of toxicity.
An alternative embodiment of the method of this invention is to analyze the
in vitro effects of pharmacologically relevant concentrations of compounds on
gene
expression in blood cells.
The Genes and Proteins Identified by the Method:
In still another embodiment, the method described above, and/or the
fingerprints generated for certain selected toxicities may be useful in
identifying
novel genes that may have a significant impact on the compound's toxicity.
Application of the compositions and methods of this invention as above
described
also provides other compositions, such as any isolated gene sequence which is
unusually reactive to the toxic result of one or more known toxicants.
For example, in a desirable embodiment, the methods of this invention is
useful in a clinical setting. Gene expression grids may aid in the
identification of the
mechanism underlying the occurrence of pathological reactions and toxicity in
a
minority of patients during human trials. Using human grids, gene expression
in
cells derived from patients/volunteers known to have experienced the adverse
event
in question during a clinical trial can be compared to gene expression from
those
who remained well. Ideally as described above, mRNA is obtained from cells of
the
target organ, but may also include mRNA obtained from blood cells in which
transcription can be altered, e.g., white blood cells. By comparing
hybridization
patterns for the affected patients vs. the well patients, a defined genetic
fingerprint or
genes that are differentially expressed to a significant degree may be
obtained.
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An embodiment of the invention is any gene sequence identified by the
methods described therein. These gene sequences associated with the toxic
reaction
are used to obtain full-length cDNA clones by conventional methods. The genes
may be studied in greater detail; e.g. through sequencing and mutation
analysis.
These gene sequences may be employed in conventional methods to produce
isolated proteins encoded thereby. To produce a protein of this invention, the
DNA
sequences of a desired gene invention or portions thereof identified by use of
the
methods of this invention are inserted into a suitable expression system. In a
preferred embodiment, a recombinant molecule or vector is constructed in which
the
polynucleotide sequence encoding the protein is operably linked to a
heterologous
expression control sequence permitting expression of the human protein.
Numerous
types of appropriate expression vectors and host cell systems are known in the
art for
mammalian (including human), insect, yeast, fungal and bacterial expression.
The transfection of these vectors into appropriate host cells, whether
I 5 mammalian, bacterial, fungal or insect, or into appropriate viruses,
results in
expression of the selected proteins. Suitable host cells, cell lines for
transfection and
viruses, as well as methods for construction and transfection of such host
cells and
viruses are well-known. Suitable methods for transfection, culture,
amplification,
screening and product production and purification are also known in the art.
In one embodiment, the essential genes and proteins encoded thereby which
have been identified by this invention can be employed as diagnostic
compositions
useful in the diagnosis of a disease or infection by conventional diagnostic
assays.
For example, a diagnostic reagent can be developed which detestably targets a
gene
sequence or protein of this invention in a biological sample of an animal.
Such a
reagent may be a complementary nucleotide sequence, an antibody (monoclonal,
recombinant or polyclonal), or a chemically derived agonist or antagonist.
Alternatively, the essential genes of this invention and proteins encoded
thereby,
fragments of the same, or complementary sequences thereto, may themselves be
used as diagnostic reagents. These reagents may optionally be detestably
labeled,
for example, with a radioisotope or colorimetric enzyme. Selection of an
appropriate diagnostic assay format and detection system is within the skill
of the art
and may readily be chosen without requiring additional explanation by resort
to the
wealth of art in the diagnostic area.
Additionally, genes and proteins identified according to this invention may
3~ be used therapeutically. For example, genes identified as essential in
accordance
with this method and proteins encoded thereby may serve as targets for the
screening
and development of natural or synthetic chemical compounds which have utility
as
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therapeutic drugs for the treatment of disease states associated with exposure
to
environmental stimuli. As an example, a compound capable of binding to a
protein
encoded by an essential gene thus preventing its biological activity may be
useful as
a drug component preventing diseases or disorders resulting from exposure of
the
mammalian cells to the environmental stimuli. Alternatively, compounds which
inhibit expression of an essential gene are also believed to be useful
therapeutically.
In addition, compounds which enhance the expression of genes essential to the
growth of an organism may also be used to promote the growth of a particular
organism.
Conventional assays and techniques may be used for screening and
development of such drugs. For example, a method for identifying compounds
which specifically bind to or inhibit proteins encoded by these gene sequences
can
include simply the steps of contacting a selected protein or gene product with
a test
compound to permit binding of the test compound to the protein; and
determining
the amount of test compound, if any, which is bound to the protein. Such a
method
may involve the incubation of the test compound and the protein immobilized on
a
solid support. Still other conventional methods of drug screening can involve
employing a suitable computer program to determine compounds having similar or
complementary structure to that of the gene product or portions thereof and
screening those compounds for competitive binding to the protein. Identical
compounds may be incorporated into an appropriate therapeutic formulation,
alone
or in combination with other active ingredients. Methods of formulating
therapeutic
compositions, as well as suitable pharmaceutical carriers, and the like are
well
known to those of skill in the art.
Accordingly, through use of such methods, the present invention is believed
to provide compounds capable of interacting with these genes, or encoded
proteins
or fragments thereof, and either enhancing or decreasing the biological
activity, as
desired. Thus, these compounds are also encompassed by this invention:
All publications, including but not limited to patents and patent
applications,
cited in this specification are herein incorporated by reference as if each
individual
publication were specifically and individually indicated to be incorporated by
reference herein as though fully set forth.
Numerous modifications and variations of the present invention are included
in the above-identified specification and are expected to be obvious to one of
skill in
3 ~ the art. Such modifications and alterations to the compositions and
processes of the
present invention are believed to be encompassed in the scope of the claims
appended hereto.
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wo r~n~o9o Pcrics9s~o~as
The invention is illustrated by the following examples.
Examples
Gene expression measurements using Microarrays
Source of cloned sequences
Sequences were derived from several sources. IMAGE clones (human derived
cDNA sequences inserted into bacterial plasmids) were ordered from Research
Genetics in duplicate. The stocks were streaked out onto agar plates, and 3
colonies
per clone were PCR screened with gene specific primers to determine which
clones
contained the correct sequences. Positive clones were then sequenced (ABI
automated sequencer) and checked against the sequence database to ensure the
clones were correct. Six clones were prepared de novo by PCR from SB human
cDNA. Rat, mouse and dog clones were prepared de novo by Reverse
Transcriptase-PCR (RT-PCR) from species specific RNAs using gene specific
primers and were also sequence confirmed. Stocks containing the correct clones
were preserved as glycerol stocks. In total the microarray comprises of: 77
sequences representing 45 different mammalian genes; and 5 yeast gene
sequences.
Preparation of DNA for the microarray
DNA was amplified in 96 well plates on a Perkin Elmer 9600 Thermal Cycler
using
a mixture of vector primers specific for BSK and pT7T3 (Pharmacia). Total
reaction
volume was 100u1 containing the following: lul of culture from the stock
containing
the correct clone, l0ul lOX PCR buffer (10X=300mM Tricine, 20mM Magnesium
Chloride, SOmM BetaMercaptoEthanol), O.SuI Perkin Elmer Taq polymerase
(SU/ul), 200uM dNTP's (Amersham), SOng each primer, including Universal
Forward and Reverse, as well as 2 primers made to the Pharmacia pT7T3 vector.
38 amplification cycles were carried out: 2 minutes @ 94°C initial soak
(1 cycle); 35
seconds @ 94°C (autoincrement 1 sec per cycle); 30 seconds @
55°C; 1 minute 45
seconds @ 72°C (autoincrement 1 sec per cycle) and a 10 minutes @
72°C final
extension period.
PCR yields and specificity were checked by agarose gels, and the products were
Ethanol precipitated as follows, in Nunc 96 well V-bottom plates. 10u1 of 3M
Sodium Acetate was added to the 100u1 PCR reaction, mixed, then 275u1 of 100%
Ethanol was added, and mixed again. Plates were stored at -20°C for 20
minutes,
followed by a 30 minute spin in a Beckman GS-6R tabletop centrifuge using
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Beckman Microplus carriers, at 3000rpm, 4°C. Pellets were visible at
the bottom of
the wells, which were washed with SOuI 70% Ethanol, and spun again at 3000rpm
for 20 minutes. Pellets were air dried, and resuspended at 300ng/ul in
distilled
water.
Preparation of the Microarray
A 10 ul aliquot from each of the suspended PCR products was mixed with an
equal
volume of 11M NaSCN (J. T. Baker) and deposited into individual wells of 96-
well
microtiter plates (Nunc). Approximately 1 nl of each sample was arrayed in
duplicate onto silanized (3-aminopropyl trimethoxy silane treated) glass
slides using
high-speed robotics (Molecular Dynamics Generation II Microarray System). The
average diameter of each array element was measured at 215 microns with the
spot-
to-spot centers at a distance of 500 microns. After printing, the slides were
allowed
to air dry and then placed into a vacuum oven for 2 hours at 80°C.
Prior to
hybridization, the slides were washed for 10 minutes in isopropanol, boiled
for 5
minutes in ddH20, and air dried.
Preparation of cDNA probes
Probes were prepared by simultaneous reverse transcription and labelling in
the
presence of a fluorophore. The reactions were carried out with a GibcoBRL
Superscript IIT"' kit (Preamplification System for First Strand cDNA
Synthesis) and
the protocol was as follows:
1 Oug of QuiagenTM cleaned sample RNA was mixed with tug of anchored oligo
dT20 (Cambio} in DEPC treated water to a final volume of 11.2u1. The mix was
heated to 68°C for 10 minutes and returned to ice for 1 minute.
A PCR reaction mix was prepared and kept on ice until required: 2u1 X10 PCR
buffer (supplied with kit), 2u125mM MgCl2, lul dNTP mix (to give SOOuM final
concentration of each of dATP, dGTP and dTTP, and a final concentration of
280uM
of dCTP), 0.8u1 Cy3TM dCTP (Amersham) to give a final concentration of 40uM
and
2u10.1M DTT to give a total volume of 7.8u1.
The annealed RNA (1 l.2ul) was added, on ice, to the 7.8u1 PCR reaction mix,
mixed
gently and then incubated at 39.5°C for 5 minutes. lul of Superscript
IITM (200U/ul)
was added, mixed gently, and the mix incubated at 39.5°C for a further
60 minutes.
A further lul of Superscript IIT"' was added and incubated at 39.5°C
for another 60
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minutes. The reaction was terminated by heat inactivating the Superscript II
at 68°C
for 5 minutes.
RnaseH (2U/ul) was added and incubated at 39.5°C for 20 minutes and
the probe
cleaned up by running through a QuiaquickTM PCR column according to the
manufacturers instructions.
Yeast control RNA's were made by in vitro transcription of cloned YGL097,
YDR432, YML 113, YFL021 and YGRO 14 cDNA's using a Riboprobe in vitro
Transcription System (Promega). For quality assurance purposes, the yeast
RNA's
were added to the reaction at ratio's of 1:100, 1:1,000, 1:5,000, 1:10,000 and
1:20,000 (wt/wt) respectively. After incubating the reaction at 39.5°C
for 60
minutes, an additional lul of Superscript II RT was added and incubated at
39.5°C
for a further 120 minutes. Following termination of the reaction, 1 ul of
RNase A
(l0ug/ul) and lul of RNase H were added and incubated at 39.5°C for 20
minutes.
Unincorporated label was removed by passing the reaction down a Qiaquick PCR
Purification Kit (Qiagen) according to the manufacturers protocol. To ensure
the
probe was completely free of unincorporated nucleotide, the above procedure
was
repeated before drying the probe to completion in vacuo.
Hybridisation
The probe was dried down and resuspended in l2ul (for full-length cover slips)
or
4ul (for small cover slips) of hybridisation buffer (SxSSC, 0.1% SDS, 0.25uM
pA20) and incubated at 100°C for 5 minutes. The probe mixture was
pipetted onto
the microarray surface and covered with a glass cover slip and sealed with
latex
glue. The microarray was transferred to a hybridisation oven and incubated at
42°C
for 15 hours.
Washing
The glue and coverslip was removed whilst the microarray slide was immersed in
a
bath of low stringency buffer (2xSSC, 0.1% SDS) at room temperature and the
slide
incubated for 5 minutes. The slide was then washed in a high stringency wash
(O.SxSSC, 0.1% SDS) on a flat bed shaker at room temperature for 5 minutes.
After
repeating the high stringency wash, the microarray slide was quicky placed in
a
SOmI Falcon tube and centrifuged (2 minutes at 200 x g) to remove any traces
of
wash buffer.
Data Capture
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Fluorescence from the microarray was detected and quantitated using a
Molecular
Dynamics Gen II scanner. The fluorescent signal is measured as intesity per
mm2.
A background measurement for each spot was taken in an area surrounding each
spot.
Analysis of Data
Gene Expression analysis from microarrays
After background subtraction the density for each spot was "normalised" by
calculating the ratio of the spot density to the sum of all the spot densities
and
expressed as the nDxA (for normalised density per unit area). The ratio (T/C)
of the
treated vs control values was calculated for each spot for each treatment and
time
point. This was done for spot set l and spot set 2 separately. Starting with
spot set 1
sequences having T/C ratios of >2 and <0.5 were identified as showing
differential
gene expression. If the signal was weak (< 0.35) in both spot sets for both
treated
and control samples, that sample was removed from the analysis as being
outside the
detectable range. The spot images of each of the identified sequences were
examined for dust spots or other "noise" which would give an incorrect
densitometric value. Each differentially expressed sequence was ranked
according
to fold increase/decrease.
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