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WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 



PCT 

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent Classification 6 : 

C12N 15/12, C12Q 1/68, C07K 14/47 



Al 



(11) International Publication Number: 
(43) International Publication Date: 



WO 99/27090 

3 June 1999 (03.06.99) 



(21) International Application Number: PCT/GB98/03445 

(22) International Filing Date: 20 November 1998 (20.1 1.98) 



(30) Priority Data: 

60/066,241 



20 November 1997 (20.11.97) US 



(71) Applicants (for all designated States except US): SMITHK- 

LINE BEECHAM CORPORATION [US/US]; One 
Franklin Plaza, Philadelphia, PA 19101 (US). SMITHK- 
LINE BEECHAM PLC [GB/GB]; "New Horizons Court, 
Brentford, Middlesex TW8 9EP (GB). 

(72) Inventors; and 

(75) Inventors/Applicants (for US only): BERTRAM, Timothy 
[GB/GB]; SmithKline Beecham Pharmaceuticals, The Fry- 
the, Welwyn, Hertfordshire AL6 9AR (GB). BROWNE, 
Michael, J. [GB/GB]; SmithKline Beecham Pharmaceuti- 
cals, New Frontiers Science Park South, Third Avenue, Har- 
low, Essex CM19 SAW (GB). BUGELSKI, Peter [US/GB]; 
SmithKline Beecham Pharmaceuticals, The Frythe, Welwyn, 
Hertfordshire AL6 9AR (GB). ENGLAND, Paul [GB/GB]; 
SmithKline Beecham Pharmaceuticals, New Frontiers Sci- 
ence Park South, Third Avenue, Harlow, Essex CM 19 5AW 
(GB). MITCHELL, Ian [GB/GB]; SmithKline Beecham 
Pharmaceuticals, The Frythe, Welwyn, Hertfordshire AL6 



9AR (GB). RUT, Andrew [GB/GB]; SmithKline Beecham 
Pharmaceuticals, New Frontiers Science Park South, Third 
Avenue, Harlow, Essex CM19 SAW (GB). MORGAN, 
Gwyn [GB/GB]; SmithKline Beecham Pharmaceuticals, The 
Frythe, Welwyn, Hertfordshire AL6 9AR (GB). 

(74) Agent: GIDDINGS, Peter, John; SmithKline Beecham, Corpo- 
' rate Intellectual Property, Two New Horizons Court, Brent- 
ford, Middlesex TW8 9EP (GB). 



(81) Designated States: AL, AM, AT, AU, AZ, BA, BB, BG, BR, 
BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, 
GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP, ICR, 
KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, 
MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, 
SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW, 
ARIPO patent (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), 
Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), 
European patent (AT, BE, "CH, CY, DE, DK, ES, FI, FR, 
GB, GR, IE, IT, LU, MC, NL, PT, SE), OAPI patent (BF, 
BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, 
TD, TG). 



Published 

With international search report. 



(54) Title: METHODS FOR IDENTIFYING THE TOXIC/PATHOLOGIC EFFECT OF ENVIRONMENTAL STIMULI ON GENE 
TRANSCRIPTION 



(57) Abstract 



Methods are disclosed for assessing the toxic or pathologic effects of a selected environmental stimulus or reagent on a mammalian 
cell by determining on a DNA grid a "fingerprint" hybridization pattern. The fingerprint pattern is characteristic of chemically or structurally 
diverse stimuli or reagents, which have a common adverse effect on gene transcription. A test compound is screened for a similar toxic 
effect by comparing its hybridization pattern on a similar grid to the fingerprint. 



BNSDOCID: <WO 9927090A1_L> 



• 



FOR THE PURPOSES OF INFORMATION ONLY 
Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT. 



AL 


Albania 


ES 


Spain 


LS 


Lesotho 


SI 


Slovenia 


AM 


Armenia 


FI 


Finland 


LT 


Lithuania 


SK 


Slovakia 


AT 


Austria 


FR 


France 


LU 


Luxembourg 


SN 


Senegal 


AU 


Australia 


GA 


Gabon 


LV 


. Latvia 


SZ 


Swaziland 


AZ 


Azerbaijan 


GB 


United Kingdom 


MC 


Monaco 


TD 


Chad 


BA 


Bosnia and Herzegovina 


GE 


Georgia 


MD 


Republic of Moldova 


TG 


Togo 


BB 


Barbados 


GH 


Ghana 


MG 


Madagascar 


TJ 


Tajikistan 


BE 


Belgium 


GN 


Guinea 


MK 


The former Yugoslav 


TM 


Turkmenistan 


BF 


Burkina Faso 


GR 


Greece 




Republic of Macedonia- 


TR 


Turkey 


BG 


Bulgaria 


HU 


Hungary 


ML 


Mali 


TT 


Trinidad and Tobago 


BJ 


Benin 


IE 


Ireland 


MN 


Mongolia 


UA 


Ukraine 


BR 


Brazil 


IL 


Israel 


MR 


Mauritania 


UG 


Uganda 


BY 


Belarus 


IS 


Iceland 


MW 


Malawi 


US 


United States of America 


CA 


Canada 


IT 


Italy 


MX 


Mexico 


UZ 


Uzbekistan 


CF 


Central African Republic 


JP 


Japan 


NE 


Niger 


VN 


Viet Nam 


CG 


Congo 


KE 


Kenya 


NL 


Netherlands 


YU 


Yugoslavia 


CH 


Switzerland 


KG 


Kyrgyzstan 


NO 


Norway 


ZW 


Zimbabwe 


CI 


Cdte d'lvoire 


KP 


Democratic People's 


NZ 


New Zealand 






CM 


Cameroon 




Republic of Korea 


PL 


Poland 






CN 


China 


KR 


Republic of Korea 


PT 


Portugal 






CU 


Cuba 


KZ 


Kazakstan 


RO 


Romania 






CZ 


Czech Republic 


LC 


Saint Lucia 


RU 


Russian Federation 






DE 


Germany 


LI 


Liechtenstein 


SD 


Sudan 






DK 


Denmark 


LK 


Sri Lanka 


SE 


Sweden 






EE 


Estonia 


LR 


Liberia 


SG 


Singapore 







BNSDOCID: <WO 9927090Al_i. > 



XVO 99/27090 -w PCT/GB 98/03445 



METHODS FOR IDENTIFYING THE TOXIC/PATHOLOGIC EFFECT 
OF ENVIRONMENTAL STIMULI ON GENE TRANSCRIPTION 

5 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, 
infeciious agents, etc, for a toxic or pathologic effect upon gene transcription. 

Mammalian cells frequently respond to exogenous stimuli of many types by 
10 . 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 
1 5 exposure to U V 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 
20 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 
25 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 
30 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 
35 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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WO 99/27090 PCT/GB98/03445 

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 
5 particular gene sequences on the basis of their gene products. For example, see 
International Patent Application No. W09 1/07087, published May 30, 1991. In 
addition, methods have been described for the amplification of desired sequences. 
For example, see International Patent Application No. W09 1/1 7271, published 
November 14, 1991. 

10 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 

15 of a selected environmental factor on a mammalian subj ect, 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 

20 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 RN A in said ceils; 

(c) extracting and isolating mRNA from said exposed cells, tissue or 
organ of step (b); 

25 (A) 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 

30 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 

35 transcriptional regulation of said mammalian cells, tissue or organ exposed to said 
factor. 



BNSDOC1D: <WO 9927090A1J_> 



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PCT/GB98/03445 



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 

5 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 

10 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 

15 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. 

20 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 
25 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. 
30 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 
35 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 

- 3 - 



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PCT/GB98/03445 



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 pharmaceutical 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," 



WO 99/27090 



PCT/GB98/03445 



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 
5 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 

10 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. WO91/07087, published May 30, 
1991 . Examples of other useful supports include, but are not limited to, 

15 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 

20 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 

25 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 
30 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 
35 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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PCT/GB98/03445 



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 mRNA to the DNA 
fragment (oligonucleotide) on the grid. Preferred labels are fluorescent molecules or 
radioactive molecules. A variety of well-known labels . can be used. 



WO 99/27090 



PCT/GB98/03445 



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 predefined 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 
cDN A 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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f 



WO 99/27090 



PCT/GB98/03445 



disclosed by Sambrook et al. f 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- 
5 512. 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 

1 0 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 

1 5 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., * 
20 100 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. 

25 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). 

30 ° ne desirable 1 method for attaching these materials to a solid support is 

described in International Application No. PCT/US90/06607 (published May 30, 
1 991). 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 

35 which enable selective activation of the predefined regions. Upon activatiorT, these 
binding substances become capable of binding and immobilizing the materials 
derived from the genomic library. 



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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. 
5 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. 

10 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 

15 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., UV 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 

20 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 

25 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 

30 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 

35 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. 

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 will 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 
5 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 
10 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. 
1 5 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 
20 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. 
25 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 
30 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 
35 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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10 



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 
15 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. 

20 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 
25 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 
30 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. 



12 



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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. 
5 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 
10 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 iransfection of these vectors into appropriate host cells, whether 
1 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. 
20 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 detectably targets a gene 
sequence or protein of this invention in a biological sample of an animal. Such a 
25 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 detectably labeled, 
30 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 
35 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 
5 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. 

10 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 

15 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 

20 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. 

25 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, 

30 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 

35 the art. Such modifications and alterations to the compositions and processes of the 
present invention are believed to be encompassed in the sco^e of the claims 
appended hereto. 



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The invention is illustrated by the following examples. 
Examples 

Gene expression measurements using Microarrays 
Source of cloned sequences 

5 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 

10 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 

15 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 DN A for the microarray 

DN A 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 

20 volume was lOOul containing the following: lul of culture from the stock containing 
the correct clone, lOul 10X PCR buffer (10X=300mM Tricine, 20mM Magnesium 
Chloride, 50mM BetaMercaptoEthanol), 0.5ul Perkin Elmer Taq polymerase 
(5U/ul), 200uM dNTP's (Amersham), 50ng each primer, including Universal 
Forward and Reverse, as well as 2 primers made to the Pharmacia pT7T3 vector. 

25 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 
30 Ethanol precipitated as follows, in Nunc 96 well V-bottom plates. 1 Oul of 3M 

Sodium Acetate was added to the lOOul PCR reaction, mixed, then 275ul 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 50ul 70% Ethanol, and spun again at 3000rpm 
for 20 minutes. Pellets were air dried, and resuspended at 300ng/ul in distilled 
water. 

5 Preparation of the Microarray 

A 1 0 ul aliquot from each of the suspended PCR products was mixed with an equal 
volume of 1 1M 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 

1 0 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 

15 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 II™ kit (Preamplification System for First Strand cDNA Synthesis) and 
20 the protocol was as follows: 

lOug of Quiagen™ cleaned sample RNA was mixed with 2ug of anchored oligo 
dT 20 (Cambio) in DEPC treated water to a final volume of 1 1 .2ul. 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: 2ul XI 0 PCR 
25 buffer (supplied with kit), 2ul 25mM MgCl 2 , lul dNTP mix (to give 500uM final 

concentration of each of dATP, dGTP and dTTP, and a final concentration of 280uM 
of dCTP), 0.8ul Cy3™ dCTP (Amersham) to give a final concentration of 40uM and 
2ul 0. 1 M DTT to give a total volume of 7.8ul. 

The annealed RNA (1 1.2ul) was added, on ice, to the 7.8ul PCR reaction mix, mixed 
30 gently and then incubated at 39.5°C for 5 minutes. 1 ul of Superscript II™ (200U/ul) 
was added, mixed gently, and the mix incubated at 39.5°C for a further 60 minutes. 
A further lul of Superscript II™ 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 Quiaquick™ PCR column according to the 
5 manufacturers instructions. 

Yeast control RNA's were made by in vitro transcription of cloned YGL097, 
YDR432, YML1 13, YFL021 and YGR014 cDNA's using a Riboprobe in vitro 
Transcription System (Promega). For quality assurance purposes, the yeast RNA f s 
were added to the reaction at ratio's of 1 : 1 00, 1 : 1 ,000, 1 :5,000, 1 : 1 0,000 and 

10 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, lul of RNase A 
(lOug/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 

1 5 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 12ul (for full-length cover slips) or 
20 4ul (for small cover slips) of hybridisation buffer (5xSSC, 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 1 5 hours. 

25 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 
(0.5xSSC, 0.1% SDS) on a flat bed shaker at room temperature for 5 minutes. After 
30 repeating the high stringency wash, the microarray slide was quicky placed in a 
50ml 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 mm 2 . 
A background measurement for each spot was taken in an area surrounding each 
spot. 

5 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 

10 treated vs control values was calculated for each spot for each treatment and time 

point. This was done for spot set 1 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 

15 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. 



18 



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CLAIMS: 

1 . 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 
transcriptional regulation of said mammalian cells, tissue or organ exposed to said 
factor. 

2. The method according to claim 1 wherein said grid comprises unique nucleic 
acid sequence tags from human genes. 

3. The method according to claim 1 or 2 wherein said grid comprises unique 
nucleic acid sequence tags from genes cloned from a selected tissue or cell line. 

4. The method according to any one of the preceding claims wherein said grid 
comprises unique nucleic acid sequence tags from genes which are particularly 
relevant to the identification of a selected toxicity. 

5. The method according to any one of the preceding claims wherein said 
mammalian cells are exposed in vivo or in culture. 

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6. The method according to any one of the preceding claims wherein said 
environmental factor is a change in the diet of said mammal or a physical condition 
to which the mammal is exposed. 

7. The method according to any one of claims 1 to 5 wherein said exposing step 
comprises: 

a) administering a transgene into said mammal; 

b) eliminating a gene from said mammal; 

c) administering an exogenous compound to said mammal;. 

d) administering an endogenous compound or an analogue thereof to 
said mammal; or 

e) exposing said mammal or cell to a pathogenic microorganism. 

8. The method according to any one of the preceding claims wherein said 
transcriptional effect is an increase or decrease in mRNA transcription in the 
exposed mammalian cells, tissue or organ. 

9. The method according to any one of the preceding claims wherein said 
mammal is selected from a non-human primate, a rodent, a canine, and a human. 

10. The method according to anyone of the preceding claims wherein said 
detectable label is a fluorescent molecule. 

11. The method according to any one of the preceding claims wherein said 
defined gene sequences are known genes or fragments thereof. 

12. The method according to any one of the preceding claims wherein said 
defined gene sequences are unknown genes or fragments thereof. 

13. A method of predicting the toxic effect of a selected test compound on a 
mammalian subject, said method comprising the steps of: 

(a) performing the method of claim 1 by calibrating the grids with a 
plurality of known and structurally distinct toxicants having a common known toxic 
effect on mammalian subjects and generating a common "fingerprint" hybridization 
pattern characteristic of said common toxic effect; 

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WO 99/27090 PCT/GB 98/0344 5 

. •• 

(b) screening a test compound according to the method of claim 1 s steps 

■■■■■ 

- (a)-(f), to generate a test compound hybridization pattern; and 

(c) comparing the test hybridization compound to said fingerprint 
hybridization pattern, 

wherein substantial identity between the fingerprint and test patterns 
indicates that said test compound shares said common toxic effect. 

14. The method according to claim 13 further comprising eliminating the test 
compound from . an early stage of drug development on the basis of its hybridization 
pattern which is substantially identical to the fingerprint. 

15. An isolated gene sequence which reacts by altered transcription to exposure 
to an environmental factor, and which is identified by the method of claim 1. 

16. An isolated protein produced by expression of a gene sequence of claim 15. 

17. A therapeutic compound capable of modulating expression of the gene 
sequence of claim 15 for use in the prevention of a toxic reaction to said 
environmental factor. 

18. A therapeutic compound capable of modulating activity of a protein of claim 
16 for use in the prevention of a toxic reaction to said environmental factor or the 
treatment of the toxic reaction. 

19. A diagnostic composition useful for the diagnosis of a toxic reaction to an 
environmental factor comprising a reagent capable of detectably targeting a gene 
sequence of claim 15. 



^ -21 - 



BNSDOCID: <WO 9927090A1 J_> 



INTERNATIONAL SEARCH REPORT 



Iru .j^^onal Application No 

?cWts 98/03445 



A. CLASSIFICATION OF SUBJECT MATTER 

IPC 6 C12N15/12 C12Q1/68 C07K14/47 



According to International Patent Classification (IPC) or to both national classification and IPC 



B. FIELDS SEARCHED 



Minimum documentation searched (classification system followed by classification symbols) 

IPC 6 C12N C12Q C07K 



Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched 



Electronic data base consulted during the international search (name of data base and, where practical, search terms used) 



C. DOCUMENTS CONSIDERED TO BE RELEVANT 



Category - Citation of document, with indication, where appropriate, of the relevant passages 



WO 97 13877 A (LYNX THERAPEUTICS INC 
; MARTIN DAVID W (US)) 17 April 1997 
see whole doc. esp. claims and exp. 3. 

WO 97 22720 A (BEATTIE KENNETH LOREN) 
26 June 1997 

see whole doc. esp. claims 

WO 94 17208 A (XENOMETRIX INC ; FARR 
SPENCER B (US); MARQUE TODD D (US)) 
4 August 1994 
see whole doc. esp. claims 

-/-- 



Relevant to claim No. 



1-19 



1-19 



17,18 



| X I Furtn © r documents are listed in the continuation of box C. 



Patent family members are listed in annex. 



" Special categories of cited documents : 

"A" document defining the genera) state of the art which is not 
considered to be of particular relevance 

"E" earlier document but published on or after the international 
filing date 

"L" document which may throw doubts on priority claim(s) or 
which is cited to establish the publication date of another 
citation or other special reason (as specified) 

"O" document referring to an oral disclosure, use. exhibition or 
other means 

"P" document published prior to the international filing date but 
later than the priority date claimed 



T" later document published after the international tiling date 
or priority date and not in conflict with the application but 
cited to understand the principle or theory underlying the 
invention 

"X" document of particular relevance: the claimed invention 
cannot be considered novel or cannot be considered to 
involve an inventive step when the document is taken alone 

"Y" document of particular relevance; the claimed invention 

cannot be considered to involve an inventive step when the 
document is combined with one or more other such docu- 
ments, such combination being obvious to a person skilled 
in the art. 

"&" document member of the same patent family 



Date of the actual completion of the' international search 

8 February 1999 



Date ot mailing of the international search report ' 



15/02/1999 



Name and mailing address of the ISA 

European Patent Office. P.B. 5618 Patentlaan 2 
NL - 2280 HV Rijswijk 
Tel. (+31-70) 340-2040, Tx. 31 651 epo nl. 
Fax: (+31-70) 340-3016 



Authorized officer 



Muller, F 



Form PCT/ISA/210 (second sheet) (July 1992) 
BNSDOCID: <WO 9927O90A1_l_> 



page 1 of 2 





INTEI^TIONAL SEARCH REPORT , alional Applica , 10 n No 

|pCT/GB 98/03445 




C.(Continuation) DOCUMENTS CONSIDERED TO BE RELEVANT 






Category - 


Citation ol document, with indication.where appropriate, of the relevant passages F 


elevant to claim No. 




A 


SCHENA M ET AL: "PARALLEL HUMAN GENOME 
ANALYSIS: MICROARRAY-BASED EXPRESSION 
MONITORING OF 1000 GENES 
PROCEEDINGS OF THE' NATIONAL ACADEMY OF 
SCIENCES OF USA, 

vol. 93, no. 20, October 1996, pages 

10614-10619, XP002912238 

see whole doc esp. discussion 


1-19 




A 


CHEE M ET AL: "ACCESSING GENETIC 
INFORMATION WITH HIGH-DENSITY DNA ARRAYS" 
SCIENCE, 

vol. 274, no. 5287, 5 October 1996, pages 
610-614, XP002912239 
cited in the application 
see the whole document 


1-19 





Fofm PCT/ISA/2 10 (continuation o( second sheet) (July 1992) 



page 2 of 2 

BNSDOCID: <WO 9927090A1 J_> 



INTERNATIONAL SEARCH REPORT 

inform^^^pn patent family members 



PC 



C*l Application No 
98/03445 



Patent document 


Publication 




Patent family 




Publication 


cited in search report 


date 




members) 




date 


WO 9713877 A 


17-04-1997 


AU 


4277896 


A 


06-05-1996 




AU 


6102096 


A 


30-12-1996 




AU 


7717596 


A 


30-04-1997 




CN 


1193357 


A 


16-09-1998 




CZ 


9700866 


A 


17-09-1997 




CZ 


9703926 


A 


17-06-1998 




. EP 


0793718 


A 


10-09-1997 




EP 


0832287 


A 


01-04-1998 




FI 


971473 


A 


04-06-1997 




JP 


10507357 


T 


21-07-1998 




NO 


971644 


A 


oz-oo-iyy / 




NO 


975744 




05-02-1998 




PL 


324000 


A 


0"7 A/1 1 nftO 

2/-U4-199o 




W0 


9641011 


A 


19-12-1996 


WO 9722720 A 


26-06-1997 


AU 


1687597 


A 


14-07-1997 


WO 9417208 A 


04-08-1994 


AT 


160178 


T 


15-11-1997 




AU 


692434 


B 


. 11-06-1998 




AU 


6124394 


A 


15-08-1994 




DE 


69406772 


D 


18-12-1997 




DE 


69406772 


T 


12-03-1998 




EP 


0680517 


A 


08-11-1995 




ES 


2111289 


T 


01-03-1998 




GR 


3026129 


T 


29-05-1998 




JP 


9503121 


T 


31-03-1997 




US 


5811231 


A 


22-09-1998 



Form PCT/1SA/210 (patent family annex) (July 1992) 
BNSDOCID: <WO 9927090A1J_>