U.S. patent application number 12/494039 was filed with the patent office on 2009-10-22 for methods utilizing differential splicing events in blood cells for the detection of pathological events.
This patent application is currently assigned to ExonHit Therapeutics SA. Invention is credited to Laurent Bracco, Fabien Schweighoffer, Bruno Tocque.
Application Number | 20090264301 12/494039 |
Document ID | / |
Family ID | 9549896 |
Filed Date | 2009-10-22 |
United States Patent
Application |
20090264301 |
Kind Code |
A1 |
Tocque; Bruno ; et
al. |
October 22, 2009 |
METHODS UTILIZING DIFFERENTIAL SPLICING EVENTS IN BLOOD CELLS FOR
THE DETECTION OF PATHOLOGICAL EVENTS
Abstract
The present invention concerns new compositions and methods for
the detection of pathological events. It more specifically concerns
methods for the detection in vitro of the presence of a pathology
or a pathological event in a subject, comprising taking a sample of
blood cells from the subject and determining, in this sample, the
presence of blood cells presenting a physiological state
characteristic of the pathology. The invention also concerns the
tools, kits and compositions for the implementation of such
methods, as well as their uses in the field of human and animal
health, or in experimental research for example.
Inventors: |
Tocque; Bruno; (Courbevoie,
FR) ; Bracco; Laurent; (Paris, FR) ;
Schweighoffer; Fabien; (Vincennes, FR) |
Correspondence
Address: |
CLARK & ELBING LLP
101 FEDERAL STREET
BOSTON
MA
02110
US
|
Assignee: |
ExonHit Therapeutics SA
Paris
FR
|
Family ID: |
9549896 |
Appl. No.: |
12/494039 |
Filed: |
June 29, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10070297 |
Mar 5, 2002 |
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PCT/FR00/02439 |
Sep 5, 2000 |
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12494039 |
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09456461 |
Dec 8, 1999 |
6372432 |
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10070297 |
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Current U.S.
Class: |
506/7 |
Current CPC
Class: |
G01N 33/56972 20130101;
C12Q 1/6809 20130101 |
Class at
Publication: |
506/7 |
International
Class: |
C40B 30/00 20060101
C40B030/00 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 16, 1999 |
FR |
99/11563 |
Claims
1. A method for in vitro detection of a given, predefined
pathological condition associated with deregulation of a cell
signaling pathway in a human subject, wherein said given,
predefined pathological condition causes disease in a tissue
distinct from nucleated blood cells of said human subject, said
method comprising: (i) providing a sample comprising nucleated
blood cells from the subject being tested for the presence of said
pathological condition, (ii) preparing nucleic acid molecules from
the sample of step (i), (iii) hybridizing all or part of the
nucleic acid molecules from step (ii) to at least one nucleic acid
library capable of detecting the presence of said given, predefined
pathological condition when contacted with a diverse population of
nucleic acid molecules prepared from nucleated blood cells from a
human subject having said given, predefined pathological condition
under conditions allowing hybridization to occur, said library
having an ordered arrangement on a support and comprising a
plurality of nucleic acid molecules that specifically hybridize to
differentially spliced ribonucleic acid molecules (RNAs) expressed
in nucleated blood cells from human subjects known to have said
given, predefined pathological condition, wherein said
differentially spliced RNAs are characteristic of said given,
predefined pathological condition that causes disease in a tissue
distinct from nucleated blood cells, and (iv) detecting
hybridization between a plurality of said nucleic acid molecules of
said subject being tested and said nucleic acid molecules of said
library, wherein said hybridization allows determination of the
presence or absence of said given, predefined pathological
condition in said subject being tested.
2. The method of claim 1, wherein the nucleic acid molecules
prepared from the sample are total or messenger RNA or
complementary deoxyribonucleic acid (cDNA) derived therefrom.
3. The method of claim 2, wherein the nucleic acid molecules
prepared from the sample are amplified.
4. The method of claim 1, wherein the nucleic acid molecules
prepared from the sample are labeled.
5. The method of claim 1, for the detection in vitro of the stage
of progression of said given, predefined pathological condition in
said subject.
6. The method of claim 1, wherein said support is a membrane, a
glass plate, or a biochip.
7. The method of claim 1, wherein said pathological condition is
characterized by excessive cell proliferation.
8. The method of claim 1, wherein said nucleated blood cells
comprise lymphocytes, macrophages, monocytes or dendritic
cells.
9. The method of claim 1, wherein said nucleic acid library further
comprises a control nucleic acid molecule.
10. A method for in vitro detection of a given, predefined
pathological condition characterized by excessive cell
proliferation in a human subject, wherein said given, predefined
pathological condition causes disease in a tissue distinct from
nucleated blood cells of said human subject, said method
comprising: (i) providing a sample comprising nucleated blood cells
from the subject being tested for the presence of said pathological
condition (ii) preparing nucleic acid molecules from the sample of
step (i), (iii) hybridizing all or part of the nucleic acid
molecules from step (ii) to at least one nucleic acid library
capable of detecting the presence of said given, predefined
pathological condition when contacted with a diverse population of
nucleic acid molecules prepared from nucleated blood cells from a
human subject having said given, predefined pathological condition
under conditions allowing hybridization to occur, said library
having an ordered arrangement on a support and comprising a
plurality of nucleic acid molecules that specifically hybridize to
differentially spliced ribonucleic acid molecules (RNAs) expressed
in nucleated blood cells from human subjects known to have said
given, predefined pathological condition, wherein said
differentially spliced RNAs are characteristic of said given,
predefined pathological condition that causes disease in a tissue
distinct from nucleated blood cells, and (iv) detecting
hybridization between a plurality of said nucleic acid molecules of
said subject being tested and said nucleic acid molecules of said
nucleic acid library, wherein said hybridization allows
determination of the presence or absence of said given, predefined
pathological condition in said subject being tested.
11. The method of claim 10, wherein said given, predefined
pathological condition is stenosis.
12. The method of claim 10, wherein said nucleated blood cells
comprise lymphocytes, macrophages, monocytes or dendritic
cells.
13. The method of claim 10, wherein said nucleic acid library
further comprises a control nucleic acid molecule.
Description
[0001] The present invention concerns new compositions and methods
for the detection of pathological events. It more particularly
concerns compositions and methods for the remote detection of
pathological events. The invention also provides for the tools,
kits and compositions for the implementation of such methods, as
well as their uses in the field of human or animal health, or, for
example, in experimental research.
[0002] The ageing of populations in industrialized countries has
given rise to new needs in diagnostics. Diseases such as cancer or
neurodegenerative disorders would be better managed to the benefit
of both patients and society if diagnostic tools were available to
predict the onset or progression of the disease.
[0003] Experience has shown that the earlier the diagnosis is made,
the greater the chance of controlling the probable course of the
disease. This has been very clearly established in the case of
cancer. Early detection campaigns for breast cancer through the use
of routine mammography have improved the life expectancy of these
cancers. Likewise, it might be presumed that early intervention in
patients who develop Alzheimer's disease would make it possible to
significantly slow its progression.
[0004] The incidence of diseases such as cancer and
neurodegenerative disorders increases sharply with the age of the
population. It is likely that these diseases take years to develop
to the point where they can be detected. It has been found, for
example, that an accumulation of successive mutations in the human
genome is required to initiate a cancer. Similarly, genetic studies
in selected cohorts with a high incidence of Alzheimer's disease,
together with genetic experiments in animals, further underscore
the multifactorial nature of initiation of this disease.
[0005] These age-related diseases share some common features
including:
[0006] cellular alterations occurring in response to a
disequilibrium in the environment of the affected tissues due to
damage by physical, chemical or biological agents;
[0007] the involvement of cells of the immune system.
[0008] Although alterations in the affected tissues are
preferentially identified only by biopsy, it may be the case that
alterations in immune cells, which reflect an ongoing disease
process, can be detected far from the sites of development of these
diseases, since most cells in the immune system circulate between
tissues and the blood or lymph compartments.
[0009] Lymphocytic cells and macrophages are the principal
mediators of the cellular immune response. Lymphocytes and
macrophages are present in both tissues and blood. They are the
first cells to come into contact with foreign tissues. Macrophages
degrade the concerned tissues and substances. The peptides derived
from the degraded proteins are then bound by the major
histocompatibility system class II molecules which transport them
to the macrophage surface, where the complexes are recognized by T
lymphocytes. Other systems of peptide presentation and immune
response activation exist and have been described notably in the
case of development of cancer.
[0010] Today, diseases such as these are diagnosed after the
pathology is already present. In the case of cancer, for example,
the diagnosis is made on the basis of medical imaging studies and
morphological diagnosis of biopsied tissues. For diseases such as
Alzheimer's disease, the diagnosis is made on the basis of a body
of medical findings.
[0011] Thus there is a real need for tools and methods enabling
early, simple and reliable detection of the development of disease,
particularly diseases related to defects in cell signalling
regulatory mechanisms, especially those diseases characterized by
excessive cell proliferation such as cancer, neurodegenerative
disorders, stenosis, etc.
[0012] The advent of molecular biology methods combined with
bio-information technologies has made it possible to construct
libraries (or banks) of DNA fragments characteristic of a given
pathology, enabling the detection of the presence or absence of
pathological markers in a very small sample of any tissue.
[0013] The present invention now sets forth a new approach for the
detection of pathologies in vitro. More specifically, the present
invention describes new methods and compositions for the detection
of pathological events, notably pathological genetic signatures.
The invention further describes methods and compositions usable for
the remote detection of pathological events, i.e. using biological
materials distinct from the pathological tissues. The compositions
and methods provided for by the invention now offer clinicians,
biologists and industrialists new solutions for in vitro diagnosis,
based on direct, rapid, sensitive and economical methods that can
be automated.
[0014] More particularly, the present invention is based notably on
the demonstration that it is possible to determine, from biological
samples comprising circulating cells, the presence or the risk of
development of a pathology. More particularly, the invention is
based on the demonstration that it is possible to detect, in a
biological sample comprising blood cells, the existence of a
pathology, including at very early stages of initiation and
development, for which all other existing diagnostics would be
ineffective.
[0015] A first subject of the invention is based more specifically
on a process for the detection in vitro of the presence of a
pathology in a subject, comprising the taking of a sample of blood
cells from the subject and the determination, in this sample, of
the presence of blood cells displaying a physiological state
characteristic of the disease.
[0016] In a specific embodiment, the process of the invention
comprises the determination of the presence, in the sample, of
blood cells presenting a protein or a protein domain characteristic
of the disease. In this context, the term "presenting" refers to
both the presence of this protein or protein domain inside the cell
(in any cellular compartment) and its presence in the cell membrane
or at the cell surface. In this embodiment, the presence of the
protein (or protein domain) can be determined by means of
antibodies (or fragments or derivatives of antibodies) or by any
other method familiar to those skilled in the art.
[0017] In another specific embodiment, the process of the invention
comprises the determination, in the sample, of the presence of
blood cells presenting a genetic profile characteristic of the
disease.
[0018] Even more preferably, the process of the invention comprises
the determination, in the sample, of the presence of blood cells
presenting alterations in gene expression characteristic of the
presence of the disease.
[0019] Thus, the present invention is based firstly, on the use of
blood cells in a remote test for the presence of a pathological
event and, secondly, on the use of genomic methods of detection of
alterations in the expression (particularly the transcription) of
the genome in these cells.
[0020] In a specific variant, the present invention therefore
comprises more preferably the determination, in a biological
sample, of the presence of blood cells presenting transcriptional
and/or post-transcriptional alterations in gene expression
characteristic of the presence of a disease.
[0021] The invention is based on the demonstration that it is
possible to detect a pathology in the progress of development from
the genomic signatures identified in the blood cells, as
pathological embryonic foci may exist in nerve tissue such as brain
or spinal cord (sites of neurodegenerative diseases) or in any
other tissue from which a cancer can develop, for example (breast,
lung, prostate, liver, bone, etc.).
[0022] The invention demonstrates in an unexpected manner that
there exist in blood cells (preferably nuclear cells such as
lymphocytes, macrophages, monocytes, dendritic cells, etc.)
transcriptional and post-transcriptional alterations in gene
expression resulting from direct or indirect interaction(s) with
the cells during initiation of the disease.
[0023] More particularly, the invention demonstrates in an
unexpected manner that there exist in blood cells (preferably
nuclear cells such as lymphocytes, macrophages, monocytes,
dendritic cells, etc.) qualitative alterations in gene
transcription following direct or indirect interaction(s) with the
cells during initiation of the disease.
[0024] According to another preferred embodiment, the process of
the invention comprises (i) the preparation of nucleic acids from
the sample and (ii) the hybridization of the nucleic acids so
prepared with at least one bank of nucleic acids characteristics of
a pathological state, the hybridization profile indicating the
presence, in the sample, of blood cells characteristic of the
pathology.
[0025] More particularly, in one embodiment of the invention, the
bank used comprises nucleic acids specific for genes whose level of
expression is modified in a blood cell from a body in a
pathological situation.
[0026] In another embodiment of the invention, the bank used
comprises nucleic acids specific for splicing forms of genes
characteristic of a blood cell from a body in a pathological
situation.
[0027] The invention is based notably on an original method, the
qualitative analysis of differences related to the presence of
Insertions or deletions (alternative splicing) in regions essential
for the function of the gene products. These insertions and
deletions are precisely regulated and are characteristic of
physiological and pathophysiological states (especially
proliferative and differentiated states) in the cells of the body.
This level of regulation is modified during the onset, maintenance
and development of a large number of pathologies. In a preferred
embodiment, the invention is therefore also based on the
application of a genomic technology designed to routinely analyze
these deregulations for the purpose of developing predictive
diagnostic tests. The invention thus enables the identification of
the deregulated genes in circulating cells during pathological
events, and the use of these qualitative genetic events in
diagnostic tests for the prediction or detection of pathological
events, which contribute to the overall control of health
costs.
[0028] With a view to identifying the specific markers of gene
expression present in the blood cells of an organism with a
disease, for example at a stage too early to be diagnosed through
clinical examinations or classical diagnostic tests, the present
invention advantageously provides for the identification of
post-transcriptional alterations. In fact, these alterations result
primarily from a modification in the regulation of a key step in
gene expression: splicing. Alternative splicing qualitatively
modifies RNA by including or excluding exons or introns from this
RNA whose presence or absence related to a given pathophysiological
situation can provide the basis for a diagnosis. This diagnosis can
be based on the use of PCR or hybridization to allow specific
differential detection of the spliced sequence between the two
situations. Alternative splicing, through the use of alternative
exon(s) or by retention of intron(s) in a messenger RNA, often
affects the sequence of the corresponding protein. These
differences in the amino acid sequence make it possible to envision
a diagnosis based on the use of antibodies that specifically
recognize the alternative protein sequence.
[0029] As noted above, the process of the invention is based
particularly on the use of circulating cells as biological
material. More specifically, these are blood cells, preferably
nuclear cells. Lymphocytes, macrophages, monocytes, dendritic
cells, etc. may be cited in particular. These cells can be
harvested from a subject by any method known to those skilled in
the art, including cytapheresis, Ficoll gradients, preparation of
peripheral blood mononuclear cells, etc. For purposes of
implementation of the present invention, the different populations
of blood cells can be separated from each other so that only a
specific type presenting a specific genomic signature is used.
However, the test set forth by the invention can also be carried
out on a biological sample comprising unseparated blood cells.
Furthermore, the circulating cells can also be (or comprise) tumor
cells detached from the pathological tissue, for example in the
case of metastatic processes. The nucleic acids can be prepared
from the sample by any method familiar to those skilled in the art,
including cell lysis, extraction, RNA isolation, etc. Furthermore,
these nucleic acids are preferably treated prior to the
hybridization step, for example to produce cDNA, to amplify these
nucleic acids, to label them, etc. In this regard, the labelling
can be radioactive, enzymatic, fluorescent, colorimetric or of any
other type. The process of the invention typically comprises taking
a biological blood sample, treating the blood cells to release the
nucleic acids, amplifying the nucleic acids (and their reverse
transcription, as the case may be), labelling the nucleic acids and
hybridizing them on one or more banks.
[0030] The process of the invention can be used to detect the
presence of a pathology (or a pathological event), i.e. the
existence of cell mechanisms characteristic of a situation of
initiation or development of a pathology, even if the clinical
symptoms of such are not yet apparent. In this regard, the process
of the invention can also enable the detection in vitro of the
stage of progression of a pathology in a subject. Thus, the genetic
signatures of the cells evolve according to the stage of
progression of the pathology, and it is possible to detect, through
the use of specific banks, the progression of a pathology.
Furthermore, the process of the invention also enables the
detection in vitro of the site of a pathology in a subject, i.e.
the tissue in which the pathological site is present, for
example.
[0031] As noted above, the process of the invention can be
implemented for the detection of different types of pathologies,
notably pathologies associated with deregulation of cell signalling
pathways. These may be pathologies related to ageing, such as
neurodegenerative disorders for example, or any other pathology
involving particularly an abnormal level of cell proliferation,
such as cancer, stenosis, etc.
[0032] In a specific embodiment, the invention concerns a process
such as defined above for the detection in vitro of the presence,
the stage of progression and/or the site of a neurodegenerative
disorder.
[0033] In another specific embodiment, the invention concerns a
process such as defined above for the detection in vitro of the
presence, the stage of progression and/or the site of a cancerous
disease. These may be different types of cancer such as, for
example, solid tumors of the liver, lungs, head and neck, melanoma,
liver, bladder, breast, etc.
[0034] The invention also provides for a process of detection in
vitro of blood cells characteristic of a pathological state,
comprising taking a sample of blood cells from a subject and
determining the presence, in this sample, of blood cells presenting
a genetic profile characteristic of a pathology.
[0035] As noted above, the invention is based in part on the
constitution and the use of banks of nucleic acids characteristic
of a pathological state. In a first embodiment, these are banks (or
preparations) of nucleic acids comprising specific nucleic acids of
genes whose level of expression is altered in a blood cell from an
organism in a pathological situation.
[0036] In another embodiment, these are banks (or preparations) of
nucleic acids comprising nucleic acids specific for splicing forms
of genes, characteristic of a blood cell from an organism in a
pathological situation.
[0037] The preparations and banks can be deposited on supports,
refined and mixed, as described below in further detail.
[0038] The invention further describes methods for the preparation
of such banks. In particular, these methods comprise (i) obtaining
an initial nucleic acid preparation from a blood cell isolated from
an organism presenting a pathology, (ii) obtaining a reference
nucleic acid preparation from a blood cell isolated from an
organism that does not present said pathology, (iii) a
hybridization step between said initial preparation and the
reference preparation, and recovery of the nucleic acids
characteristic of the blood cell from the organism in a
pathological situation.
[0039] The invention also describes processes of preparation of
nucleic acid banks characteristic of a stage of progression of a
pathology, comprising (i) obtaining an initial nucleic acid
preparation from a blood cell isolated from an organism presenting
a pathology at a defined stage of progression, (ii) obtaining a
reference nucleic acid preparation from a blood cell isolated from
an organism presenting said pathology at a different stage of
progression, (iii) a hybridization step between said initial
preparation and the reference preparation, and (iv) the recovery of
the nucleic acids characteristic of the blood cell from the
organism in a defined stage of progression of the pathology.
[0040] As explained below in further detail, the recovery of the
clones can comprise either the recovery of non-hybridized nucleic
acid clones, or the recovery, from the hybrids formed, of nucleic
acid clones specific for splicing forms of genes.
[0041] The invention also concerns any kit that can be used for the
implementation of a process as described above comprising a nucleic
acid bank comprising nucleic acids specific for alterations in gene
expression characteristic of blood cells of an organism in a
pathological situation.
Identification of Specific Markers of Transcriptional and
Post-transcriptional Alterations
[0042] As noted above, the invention describes processes of
preparation of nucleic acid banks characteristic of a stage of
progression of a pathology, comprising (i) obtaining an initial
nucleic acid preparation from a blood cell isolated from an
organism presenting a pathology at a defined stage of progression,
(ii) obtaining a reference nucleic acid preparation from a blood
cell isolated from an organism presenting said pathology at a
different stage of progression, (iii) a hybridization step between
said initial preparation and the reference preparation, and (iv)
the recovery of the nucleic acids characteristic of the blood cell
from the organism in a defined stage of progression of the
pathology.
[0043] The methods set forth by the invention more particularly
comprise the constitution of nucleic acid clones and banks from
RNA(s) extracted from different diseases, at different stages of
their progression, and obtained from both pathological tissues and
from blood cells whose genetic expression was affected by these
tissues. These clones and banks are advantageously obtained by
methods for differential analysis of gene expression. The
differential signatures obtained are therefore specific to the
differences between the healthy tissue and the diseased tissue on
the one hand, and between the blood cells of the patient and the
blood cells of the healthy control on the other hand. These
signatures can therefore be expressed preferably in either the
pathological samples or the control samples.
[0044] The nucleic acid populations used to obtain clones or to
constitute banks are, for example, RNA (total or messenger RNA) of
cells extracted from a pathological situation and RNA (total or
messenger RNA) corresponding to a control situation, or nucleic
acids derived from this total or messenger RNA by reverse
transcription, amplification, cloning into vectors, etc. These
nucleic acids can be prepared according to methods familiar to
those skilled in the art. Briefly, these methods generally comprise
lysis of the cells, tissue or sample, and isolation of the RNA by
extraction. In particular, this can consist of a treatment with
chaotropic agents such as guanidium thiocyanate (which destroys the
cells and protects the RNA) followed by extraction of the RNA with
solvents such as phenol or chloroform. Those skilled in the art are
familiar with such methods (see Maniatis et al., Chomczynski et
al., Anal. Biochem. 162 (1987) 156), which can be easily
implemented by using commercially available kits such as the
US73750 kit (Amersham) for total RNA. The RNA used does not have to
be perfectly pure, and in particular the presence in the
preparation of traces of genomic DNA or other cellular components
such as protein, etc. is not a problem so long as they do not
significantly affect RNA stability. Furthermore, in an optional
manner, it is possible to use preparations of messenger RNA in
place of total RNA preparations. The messenger RNA can be isolated
either directly from the biological sample or from the total RNA by
means of polyT sequences according to conventional methods. In this
regard, messenger RNA can be obtained through the use of
commercially available kits such as the US72700 kit (Amersham). The
RNA can also be obtained directly from banks or from other samples
prepared in advance and/or available in collections, and stored
under suitable conditions.
[0045] The hybridization can be carried out under different
conditions which can be adjusted by those skilled in the art. The
hybridization preferably uses an excess of the nucleic acid
population derived from the deregulated situation relative to the
nucleic acid population derived from the control situation.
[0046] Using the product of the hybridization reaction, two main
types of approaches can be used to isolate the clones
characteristic of deregulation (pathological) according to the
invention. The first, which is strictly quantitative, enables the
generation of a nucleic acid preparation comprising all (or a
significant part) of the clones resulting from a difference in the
level of expression between the two situations. Such clones (and
banks) are obtained by known subtractive hybridization methods
consisting primarily of eliminating the hybrids formed during the
hybridization step and keeping only non-hybridized clones
characteristic of the deregulated situation relative to the chosen
control situation.
[0047] In a preferred embodiment, however, a qualitative process is
used to enable generation of a nucleic acid preparation comprising
all (or a large part) of the clones resulting from functional gene
mutations characteristic of the deregulated situation relative to
the chosen control situation. More particularly, such a qualitative
bank comprises not the entire group of clones whose expression is
modified, but for example clones corresponding to splicing or
deletion events that differ between the two situations. Considering
the role of alternative splicing in cell regulatory and
transformation pathways, such preparations (and banks)
advantageously comprise clones having an important functional value
and therefore likely to reflect the genetic modifications involved
in the situation of deregulation. Such clones therefore enable the
constitution of banks with greater predictive power and the
generation of more representative genetic markers.
Such qualitative banks can be constituted by the isolation from the
hybrids formed during the hybridization step of nucleic acid
regions corresponding to differential splicing or to deletions.
Depending on the methods used, these regions correspond either to
unpaired regions or to paired regions.
[0048] These two approaches are described below in more detail.
Production and use of Differential Quantitative Banks
[0049] In a first embodiment, the present invention therefore makes
use of a differential quantitative bank, i.e. a bank comprising
nucleic acid clones corresponding to genes whose level of
expression is modified in cells in the pathological situation
relative to a control situation. Such banks can also be derived
from differential quantitative analyses, pooling sequences whose
expression is increased or decreased in cellular deregulation
phenomena. The methods to establish this type of bank are familiar
to those skilled in the art and can be broken down into the
following categories:
High Flow Sequencing Electronic Subtraction
[0050] This process is based on the random sequencing of a certain
number of cDNAs. A computer search engine can then be used to
perform a subtraction between the two situations under
analysis.
Serial Analysis of Gene Expression (SAGE)
[0051] This process is based on the recognition of a signature
associated with each cDNA by using restriction enzymes and
oligonucleotide adaptors. This label corresponds to a part of the
cDNA sequence (10 nucleotides long so as to unambiguously identify
the corresponding cDNA). The labels are then assembled for
sequencing and analysis (Velculescu et al., Science, 1995, 270:
484-487). This approach therefore represents a short-cut to
systematic sequencing.
Nucleic Acid Arrays
[0052] This method is based on the application at more or less high
density of nucleic acids such as oligonucleotides, PCR fragments or
cDNAs on solid supports such as membranes, glass plates or
bio-chips. Messenger RNA probes from the healthy or pathological
samples are then used in hybridization to identify messengers that
are overexpressed or underexpressed.
Differential Display
[0053] This method makes use of an oligo-dT primer and random
primers to perform PCR on cDNA populations. The PCR products are
then compared on very high resolution gels. Differentially
expressed fragments are then isolated and their presence confirmed
by northern blot analysis prior to sequencing. Several variants of
this method have been described (Prashar and Weissman, PNAS, 1996,
93: 659-663). These variants differ in terms of the primer and
restriction enzymes and adaptor used. As with the SAGE method, they
make use of the 3'-ends of cDNAs. This approach is made accessible
by the existence of several commercially available kits.
Subtractive Cloning
[0054] This method is based on the elimination of cDNAs that are
common to the two samples under comparison. Thus, different kits in
which the "tester" cDNA is hybridized with an excess of "driver"
cDNA are available (Clontech). The final product consists of a pool
of PCR-amplified fragments derived from differentially expressed
cDNAs, which can be cloned in a suitable vector for subsequent
analysis. RDA (Representational Difference Analysis) is another
method based on this principle of subtraction (Lisitsyn et al.,
Science, 1993, 259: 946-951).
[0055] The implementation of these differential analytical methods
therefore enables the generation of quantitative banks and clones,
i.e. comprising all the sequences whose expression is increased or
decreased in cellular deregulation phenomenon or phenomena involved
in pathologies.
Production and use of Differential Qualitative Banks
[0056] In another embodiment, the present invention advantageously
uses a differential qualitative bank, i.e. a bank comprising
nucleic acid clones of which at least part of the sequence
corresponds to the sequence of the genes differentially spliced in
the pathlogical cells and the control cells. This type of bank
therefore comprises sequences that are differentially spliced in
pathological deregulatory processes.
[0057] The use of a bank of this type is particularly advantageous.
In fact, the different signalling pathways that are altered in many
diseases such as cancer and neurodegenerative disorders, for
example, involve genes and therefore mRNAs whose expression is
regulated by alternative splicing. Furthermore, a growing number of
examples furnished by the literature show that the RNA forms
specifically observed in pathological states result from
alternative splicing.
In relation to the originality of the invention, it should also be
noted that the state of activation of the different types of cells
that participate in the immune response is regulated by signalling
cascades whose mediators are regulated by splicing.
[0058] Thus, Alzheimer's disease, Huntington's disease and
Parkinson's disease are just some of the examples of diseases with
a neurodegenerative component which have a true economic impact.
Even though the description of the clinical features and the
identification of several susceptibility genes have led to
significant advances in our understanding of these diseases, the
molecular mechanisms underlying their development are still quite
obscure. The elucidation of the signalling cascades that are
deregulated in these pathological states will undoubtedly lead to
the discovery of targets amenable to diagnostic and therapeutic
measures. The literature underscores the importance of alterations
in RNA splicing processes. [0059] Spinal muscular atrophy is one of
the most common genetic diseases. Two genes, SMN1 and SMN2, encode
identical proteins. Loss of the two SMN1 alleles and a splicing
mutation in the SMN2 gene lead to disease development (Lorson et
al. Proc. Natl. Acad. Sci. USA 1999, 96: 6307-6311). [0060]
Specific splicing mutations in the presinillin gene, PS1, have been
found in the biopsy specimens of patients with Alzheimer's disease
(Isoe-Wada et al. Eur. J. Neurol., 1999: 163-167) [0061] The
glutamate transport protein is of major importance in
neurodegenerative diseases such as amyotrophic lateral sclerosis or
epilepsy, for example. Splicing mutations in this transporter
affect its function (Meyer et al. Neurosci. Left, 1998. 241:
68-70).
[0062] Many examples of inactivation of anti-oncogene activity
resulting from alternative splicing of the corresponding messengers
are now known:
[0063] In small cell lung cancer, the gene encoding the p130
protein, a member of the RB (retinoblastoma protein) family, is
mutated at a splicing consensus sequence. This mutation leads to
the elimination of exon 2 which results in an absence of protein
synthesis due to the presence of a premature stop codon. This
finding was the first to highlight the importance of RB family
proteins in tumorigenesis.
[0064] In head and neck cancers, one of the mechanisms of p53
inactivation involves a mutation in the splicing consensus
sequence.
[0065] In other types of lung cancer, the gene encoding the
p16/INK4A protein, an inhibitor of cyclin-dependent CDK4 and CDK6
kinases, is mutated at a splicing donor site. This mutation results
in the production of a truncated protein with a short half-life.
The p16 protein normally binds to CDK4 and CDK6, inhibiting their
binding to type D cyclins and, in particular, the phosphorylation
of RB, which results in an accumulation of active,
hypophosphorylated forms of RB. In the absence of p16, RB is
inactivated by phosphorylation. It should noted in fact that the
p16 locus is particularly complex and that, apart from p16
expression, it enables p19 expression through alternative splicing.
The p19 protein, which does not share any common amino acids with
the p16 protein, can bind to the MDM2 proto-oncogene and block the
cell cycle in the presence of p53, thereby acting as a tumor
suppressor.
[0066] WT1, an anti-oncogene encoding a transcriptional repressor
which, when mutated, can cause Wilms tumor, is transcribed to
several messenger RNAs generated by alternative splicing. In breast
cancer, the relative proportions of the different variants are
modified in comparison. with healthy tissue, providing diagnostic
tools and clues to understanding the importance of the different
WT1 functional domains in tumor progression.
[0067] This same phenomenon of modification of the proportions of
different messenger RNA forms and protein isoforms occurs in the
case of neurofibrin NF1 in neurofibromas;
[0068] This concept of modulation of splicing events as an
indicator of tumor progression is also strengthened by the example
of HDM2. In fact, five alternative splicing forms of HDM2 have been
detected in ovarian and pancreatic cancer, and it is especially
interesting that their expression increases with tumor stage.
[0069] LTBP, a component of the extracellular matrix of various
tissues involved in TGF-.beta. secretion and storage, is also
produced in different isoforms. One such isoform, which is probably
less sensitive to proteolysis, appears to modulate the biological
activity of TGF-.beta. and might be involved in different hepatic
pathologies.
[0070] The cellular and humoral immune responses are under
transcriptional control. The literature provides many examples of
native isoforms produced by alternative splicing involved in these
immune responses.
[0071] macrophage "scavenger" receptors are membrane glycoproteins
required for the physiological and pathological response of these
blood cells and their functions are regulated by isoforms generated
by splicing (Gough et al. J. Lipid Res; 1998; 39: 531-543.)
Activation of T lymphocytes requires the functional presence of
several regulatory proteins and receptors. Boriello et al. (J.
Immunol. 1995; 155: 5490-5497) reported the presence of isoforms of
the B7 activation cofactor, generated by alternative splicing of
this gene, thus underscoring the considerable plasticity that these
splicing variants confer to the immune response.
[0072] To take into account these phenomena and this complexity,
and to thereby isolate signatures that are specific to a
pathological state and present in blood cells, the process of the
invention advantageously makes use of splicing events
characteristic of situations of deregulation, as genetic
markers.
To do so, the present invention uses, for example, differential
qualitative nucleic acid banks produced according to "DATAS"
methodology described in the unpublished international patent
application PCT/FR 99/00547. In particular, such banks can be
prepared by hybridization between the nucleic acid population
derived from cells isolated from the blood in a pathological
situation, and the nucleic acid population derived from circulating
cells in the control situation, and isolation, from the hybrids
formed, of the nucleic acids corresponding to differential
splicing.
[0073] In this approach, hybridization is preferably carried out in
liquid phase. Furthermore, it can be carried out in any suitable
devide such as tubes (Eppendorf tubes, for example), plates or any
other suitable support commonly used in molecular biology. The
hybridization is advantageously carried out in volumes of between
10 and 1000 .mu.l, for example between 10 and 500 .mu.l. It is
understood that the device and volumes used can be easily adapted
by those skilled in the art. The quantities of nucleic acids used
for the hybridization are also known to those skilled in the art.
In general, microgram quantities of nucleic acids suffice, for
example between approximately 0.1 and 100 .mu.g. Furthermore, it is
possible to use the nucleic acids in a driver/tester ratio ranging
from approximately 50 to 0.02, preferably from 40 to 0.1. Even more
advantageously, this ratio is preferably close to or greater than
1, advantageously between approximately 1 and approximately 10. It
is understood that this ratio can be adapted by those skilled in
the art according to the conditions of the process (available
quantities of nucleic acids, physiological situations, purpose,
etc.). The other hybridization parameters, including time,
temperature and ionic strength, can also be adapted by those
skilled in the art. As a general rule, following denaturation of
the tester and driver, for example by heating, the hybridization is
carried out for approximately 2 to 24 hours at a temperature of
approximately 37.degree. C. (possibly submitted to temperature
spikes), and in standard conditions of ionic strength, which can
range from 0.1 to 5 M NaCl, for example. Ionic strength is known to
be one of the factors that determines the stringency of a
hybridization, especially in the case of hybridization on a solid
support.
According to a specific embodiment of the invention, the
hybridization is carried out in a phenol emulsion, for example
according to the PERT method ("Phenol Emulsion DNA Reassociation
Technique) described by Kohne D. E. et al. (Biochemistry, Vol. 16,
No. 24, pp 5329-5341, 1977). The hybridization is avantageously
carried out in a phenol emulsion maintained by thermocycling
(temperature increase from approximately 37.degree. C. to
approximately 60/65.degree. C.) and not by agitation, according to
the method described by Miller and Riblet (NAR 23 (1995) 2339).
[0074] Any other hybridization method in liquid phase, preferably
in emulsion, can be used within the scope of the present invention.
Furthermore, the hybridization can also be done with one of the
strands immobilized on a support. Advantageously, it is the driver
that is immobilized. This can be done notably thanks to
biotinylated primers or by any other means of immobilization known
to those skilled in the art.
[0075] Using the nucleic acid populations generated by
hybridization, the genetic markers of the invention (the clones
characteristic of qualitative genomic alterations) can be
identified by any method familiar to those skilled in the art. In
the case of RNA/DNA heteroduplex, these regions are mainly regions
of unpaired RNA (RNA loops) and can be identified and cloned by
separation of the heteroduplex and the single-stranded nucleic
acids (excess nucleic acids which did not react), selective
digestion of the double-stranded RNA (domains participating in the
heteroduplex), followed by separation of the resulting
single-stranded RNA and the single-stranded DNA. In the case of
heterotriplex, these differential splicing regions consist mainly
of regions of double-stranded DNA and can be identified and cloned
by treatment with suitable enzymes such as an enzyme that digests
RNA, followed by an enzyme that digests single-stranded DNA. The
nucleic acids so obtained are directly in the form of
double-stranded DNA and can be cloned in any suitable vector.
[0076] It is understood that other specific variants and conditions
for the isolation of nucleic acids, hybridization and obtaining of
qualitative clones, are indicated in the not-yet-published
application No. PCT/FR99/00547.
[0077] These methods enable the generation of clones and nucleic
acid banks corresponding to qualitative genetic markers which allow
blood cells from a healthy situation to be distinguished from those
from a pathological situation. As indicated in the experimental
section, these nucleic acid preparations are particularly useful
markers to diagnose neurodegenerative disorders and cancers from a
blood sample.
Diversity of the Banks
[0078] The aforementioned methods therefore enable the generation
of groups of nucleic acid clones characteristic of the differences
between a healthy and a pathological situation. Each method of
preparation generates many clones which constitute banks. These
banks can be used as is, deposited on supports, modified by the
addition or removal of clones, or different banks can be combined
or control clones added, etc.
[0079] The banks provided for by the invention can comprise 10 to
50,000 clones, more generally 10 to 10,000 clones, and even more
preferably 50 to 5,000 clones. The clones are generally deposited
in a well-ordered fashion on one or more supports so as to
facilitate analysis of the hybridization results. The support can
be composed of glass, nylon, plastic, fiber, etc. or generally be
any solid support suitable for the deposit of nucleic acids. The
banks can be deposited on the supports by conventional methods
known to those skilled in the art, as described for example in the
international application No. PCT/FR99/00547.
[0080] The banks used can comprise both the nucleic acid clones
corresponding to the genes whose level of expression is altered
(quantitative genetic markers) and the nucleic acid clones of which
at least part of the sequence corresponds to exons or introns that
are differentially spliced in a pathological and healthy situation
(qualitative genetic markers). Thus, the genetic markers can be
generated by different approaches, then pooled to obtain a response
that is as predictive as possible.
It is also possible to pool the genetic markers specifically
expressed in circulating blood cells in different pathologies
within a same bank, on a same support. Hybridization of such a bank
therefore makes it possible to monitor the development of several
pathologies using a same blood sample. A subject of the present
invention is therefore also based on a nucleic acid preparation
comprising qualitative and quantitative genetic markers
characteristic of the cellular deregulation(s) present in
circulating blood cells and indicative of pathologies. A specific
subject concerns a bank comprising genetic markers characteristic
of different situations of deregulation. Another subject of the
invention is any solid support on which at least two banks of
nucleic acids characteristic of two situations of deregulation have
been deposited. In this regard, the invention further concerns a
process of preparation of a DNA chip used to diagnose pathologies,
comprising the application, on a solid support, of one or more
nucleic acid preparations characteristic of situation(s) of
deregulation.
[0081] Furthermore, in a preferred embodiment, use is made of
nucleic acid banks refined through use by the selection of clones
based on their actual involvement in different pathologies or in
different stages of the same pathology. The initial banks can in
fact comprise all the clones characteristic of genetic events of a
situation of deregulation following onset of a pathology.
Implementation of the diagnostic process set forth by the invention
then makes it possible to observe that some of the clones hybridize
with probes from early and intermediate stages of development of
the pathology. These clones can therefore be identified as markers
of early stages and can provide a very powerful diagnostic tool in
advance of any other clinical criterion or any other diagnostic
tool.
[0082] More specifically, the present invention now describes the
identification and characterization of such clones, which can be
used as genetic markers of the presence and the progression of
diseases.
One of the major applications of the identification and cloning of
these genetic markers concerns the evaluation of the hybridizing
potential of RNA extracted from the blood cells of a given subject.
This evaluation can be carried out by hybridizing a probe
corresponding to the messenger RNA of the cells of this subject,
with one or more banks of signatures characteristic of pathological
situation(s), such as described above. This application is
described in more detail below.
Methods for Analysis and Diagnosis of Signatures of Pathologies
[0083] The invention allows determination of the presence of
signatures specific for different disease stages by hybridizing a
sample of nucleic acids from cells present in the blood
circulation, with the aforementioned genetic markers, the observed
hybridization profile indicating the pathophysiological
deregulation in the subject from which the blood sample was taken.
To this end, the genetic markers used are preferably combined into
banks so that the response can be as predictive as possible. One of
the advantages of the present invention also concerns the large
number of markers used, which makes the information obtained even
more predictive. The predictive nature of the information is
furthermore strengthened by the type of markers used and
prepared.
[0084] A specific subject of the invention is based on a method of
analysis of the status of blood cells, comprising at least one
hybridization step between a) a sample of nucleic acids from blood
cells and b) a nucleic acid bank corresponding to genetic events
characteristic of deregulation(s) in cellular signalling pathways,
the hybridization profile indicating the pathophysiological
deregulations in the organism.
[0085] Other aspects and advantages of the present invention will
emerge in the following experimental section, which should be
considered illustrative and non-limiting.
Experimental Section
Example of Neurodegenerative Disorders: ALS
[0086] Animal models give access to biological samples which can be
used to analyze the different steps in the development of a disease
and to compare these steps with healthy controls.
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease
associated with different types of inclusions such as Lewis bodies
and characterized by apoptosis of spinal and cortical motor
neurons; frontal dementia sometimes occurs before the fatal
outcome. Sporadic forms in which no mutations have been identified
coexist alongside familial forms (FALS) associated with mutations
in the
[0087] SOD1 gene encoding superoxide dismutase. Transgenic mice
expressing the human SOD1 gene bearing one of the mutations seen in
FALS (mutation G93A) are available from Jackson Laboratory provided
that a user's license is obtained from Northwestern University. The
onset of the symptoms of ALS due to the G93A mutation in SOD1 does
not result from a reduction in superoxide dismutase activity, but
from an increase in its function which enhances its capacity to
generate free radicals. This model reproduces in 120 days a disease
with a fatal outcome having a symptom profile similar to the human
disease. This model provides access to brain, spinal and peripheral
blood samples.
Identification of Specific Splicing Forms in the ALS Model:
[0088] ExonHit Therapeutics has developed an original approach of
differential qualitative screening using DATAS (Differential
Analysis of Transcripts Alternatively Spliced) is technology. This
technology is the subject of a patent application in Europe and the
United States. The sequences identified by DATAS can be derived
from alternative exons or from retention of introns in one of the
two pathophysiological situations under comparison. The resulting
data therefore characterize the modifications in the expression of
RNA sequences which affect functional domains of proteins.
Differential qualitative analysis is carried out on samples from
transgenic animals and syngeneic controls aged 60 and 100 days.
Sixty days corresponds to a stage that occurs shortly before the
onset of first symptoms, but which is already characterized by
changes in brain cell physiology, particularly by an alteration in
mitochondrial metabolism. At 100 days, 50% of cortical and spinal
motor neurons are dead and active apoptosis of neurons is triggered
in parallel to activation of astrocytes.
Differential qualitative analysis is therefore carried out: [0089]
on RNA extracted from brain and spinal cord specimens without
preliminary isolation of neurons so as to take into account a
maximum of alternative splicing events related to development of
the disease. [0090] on peripheral whole blood or blood cell
fractions.
[0091] The sequences identified by DATAS correspond to introns
and/or exons whose. differential expression via splicing between
pathological situations and the healthy situation are validated by
PCR.
Comparison of these sequences with data bases allows classification
of the resulting information and a well-grounded choice of two
sequences to be submitted to further study.
Subsequent Characterization of the Sequences Obtained:
[0092] The sequences validated by PCR can be screened in
complementary models involving neurodegenerative processes. For
example, RNA from a model of cerebral ischemia or RNA from an
animal model of a prion disease can be usefully studied to validate
the selective expression of ALS markers or more generally of
markers of neurodegenerative diseases.
The expression of the identified splicing forms will be sought in
human samples from different pathologies with a neurodegenerative
component:
[0093] blood samples from patients with neurodegenerative diseases
such as Alzheimer's disease, Parkinson's disease, etc.
Example of Cancer: Upper Gastrointestinal Tract and Respiratory
Tract Cancer
[0094] The transduction of transgenes has been the subject of a
number of experimental applications in the field of experimental
oncology. Thus, the dominant alleles of certain oncogenes are used
to obtain transgenic mice, now the experimental model for the study
of cancer.
Cancer is a heterogeneous family of diseases, each characterized by
a complex group of gene mutations resulting in abnormal cell
proliferation and dissemination of metastases. Although the
identification of the gene mutations that induce the onset and
progression of cancer now appears essential for the diagnosis and
monitoring of tumor progression, it is the prospect of developing
new, early diagnostic tools based on this knowledge that explains
the important stake this research represents for the pharmaceutical
industry. There are many genes which, when mutated, can give the
cell cancerous properties. These genes play essential roles not
only during development but also throughout the life of the cell.
For example, they carry out functions as vital as growth,
differentiation, DNA repair and cell survival. Genes which, when
mutated, lead to the production of proteins which abnormally
activate the cell cycle are called oncogenes. This category
includes the cellular genes myc and ras, for example. In contrast,
anti-oncogenes normally act to slow down the cell cycle. Inhibition
of their activity makes the cell dependent solely on genes with a
proliferative effect, therefore promoting tumor progression. This
category includes the genes RB (retinoblastoma) and p53. Alongside
the oncogenes and anti-oncogenes, the genes that modulate
programmed cell death, or apoptosis, appear to be important players
in oncogenesis. Similar to a process of physiological cell
differentiation, programmed cell death is under genetic control.
Loss of this ability to trigger terminal differentiation allowing
removal of the cell places that cell in a situation of abnormal
survival which can favor the emergence of a transformed clone. This
is what is observed in human follicular lymphomas where the gene
bcl-2 is overexpressed due to a translocation between chromosomes
14 and 18. This overexpression of an anti-apoptotic gene promotes
abnormally long survival of cell populations in which other
transforming mutations can accumulate. Programmed cell death, or
apoptosis, is currently known to be an essential mechanism by which
to eliminate cells which have become undesirable, whether due to a
viral infection or because they have accumulated mutations that
render them nonfunctional or hyperproliferative. The level of
complexity governing cellular homeostasis results not only from the
large number of players involved but also from the various roles
that each can alternately play according to cell type or
conditions. The p53 anti-oncogene and the cMyc proto-oncogene also
play an important role in apoptosis control. Such complexity
requires the use of approaches that are global enough to analyze
modulations in the expression of all the involved genes, but also
sufficiently specific so that the most relevant alterations in
terms of diagnosis, monitoring of tumor progression or
identification of new drug targets can be identified as quickly as
possible.
[0095] By using different drivers of transcription (promoter
regions), it is possible to preferentially obtain expression of the
transgene in a specific tissue. In this manner one can establish
tumor models that develop in a specific tissue environment. These
different targeted tumor models suggest that it might be possible
to detect specific signatures in circulating cells according to
tumor location.
Liver-targeted Tumor Model
[0096] The murine hepatocarcinoma (HCC) model is linked to the
restricted expression in liver of the SV40 virus early sequences
encoding the large and small T antigens (Dubois, N., Bennoun, M.,
Allemand, I., Molina, T., Grimber, G., Daudet-Monsac, M., Abelanet,
R., and Briand, P. (1991) Time course development of differentiated
hepatocarcinoma and lung metastasis in transgenic mice. J.
Hepatol., 13, 227-239). The transgene is under the control of the
human antithrombin III promoter which drives early, continuous
expression of the viral antigens. For this reason, hepatocellular
proliferation undergoes a two-step perturbation. The proliferation
index of the transgenic hepatocytes is proportionally higher than
normal during liver development (from birth to 5 weeks), and then
appreciably decreases, without however reaching the low levels
characteristic of normal, quiescent liver. These transgenic mice
systematically develop differentiated HCC that are fatal to all
animals before 7 months. Despite an early deregulation of
hepatocyte proliferation, hepatomegaly occurs only late in the
course. Analysis of the preneoplastic steps preceding development
of HCC has revealed the existence of an apoptotis compensating
mechanism that maintains normal liver mass in this model (Allemand
et al., 1995). It is noteworthy that this apoptosis stops at the
very moment when the normal liver enters quiescence. Beyond this
point, it appears that hepatic homeostasis is no longer controlled.
A systematic study of sensitivity to apoptosis showed that the
hepatocytes derived from this transgenic model had acquired
resistance to cell death which was dependent on the CD95/Fas system
(Rouquet N, Allemand I, Molina T, Bennoun M, Briand P and Joulin V.
(1995) Fas-dependent apoptosis is impaired by SV40 T-antigen in
transgenic liver. Oncogene, 11, 1061-1067
[0097] Rouquet N. Allemand I, Grimber G, Molina T, Briand P and
Joulin V. (1996) Protection of hepatocytes from Fas-mediated
apoptosis by a non-transforming SV40 T-antigen mutant. Cell Death
& Diff., 3, 91-96) by a mechanism independent of alternative
splicing of the CD95/Fas receptor. However, only a global analysis
of splicing alterations can explain an alteration in this process
for all the players involved in the CD95/Fas receptor signalling
pathway.
This transgenic model is an ideal tool to identify 1) the
modifications in gene expression accompanying preneoplastic
transition to neoplasia, whether the genes are essential for
transformation (oncogenes) or inhibit tumor progression (apoptotic
genes); 2) circulating signatures of cancer development linked to
escape of tumor cells from the tumor; 3) events of alteration of
gene expression in blood cells characteristic of development of the
cancer.
Identification of Specific Signatures
[0098] The differential qualitative approach is carried out using
RNA extracted from liver and blood cells of normal mice and of mice
that develop hepatocarcinoma (HCC) due to antithrombin III-driven
expression of the SV40 T antigen. Control and transgenic animals
are chosen at different ages so as to be able to study very early,
preneoplastic and neoplastic stages which, in this model, are
characterized in particular by an activation and then an
inactivation of apoptosis required for hepatic homeostasis.
Use of the Identified Sequences
[0099] Alterations in the expression of these sequences can then be
screened in human tumor biopsies so as to broaden the field of
application in human therapy and diagnostics.
[0100] These cDNAs are used to monitor tumor progression in this
transgenic model and in a series of transgenic murine HCC models
(Bennoun M, Grimber G, Couton D, Seye A, Molina T, Briand P and
Joulin V. (1998) The amino-terminal region of SV40 large T antigen
is sufficient to induce hepatic tumours in mice Oncogene, 17, in
press). Thus, by using specific cDNAs detected at different, very
early stages in blood cells, before development of the tumor, it is
possible to predict, in a mixed population of healthy and
transgenic mice, which animals will develop a tumor.
[0101] Nucleotide probes or PCR primers derived from these
tumor-specific cDNAs can be used to screen for the expression, in
human tumor biopsies, of the identified splicing forms and/or the
RNAs whose quantities are altered in this model. Similarly, the
probes identified in the blood of animals at different stages of
tumor development can also be used to detect signatures common to
blood samples from cancer patients.
In a strategy which uses the cDNA banks obtained according to the
aforementioned processes of the invention, a total probe prepared
from blood samples from cancer patients can also be used to screen
for signatures common to the different banks established from
murine models at different stages of tumor progression, on the one
hand, and from biopsies of different human tumors on the other
hand. These hybridizations are carried out according to methods
familiar to those skilled in the art (in particular, consult the
hybridization conditions set forth in application No
PCT/FR99/00547)
Research for Predictive Diagnosis in Man
[0102] The methods described in this section can be implemented by
using either the quantitative or the qualitative analytical methods
described above. Nonetheless, the invention favors the use and
research of markers linked to qualitative alterations in gene
expression, due to the aforementioned advantages.
[0103] The invention describes the identification and constitution
of banks of signatures characteristic of the progression of a
pathology, from biopsy samples. It is thus possible to establish
banks of cDNA sequences representative of the course and site of
these pathologies. It is therefore possible to screen blood samples
for the presence of cDNA signatures identical to those present in
the banks. The presence of common signatures then indicates the
presence of nucleic acids with the same alterations as those found
in the biopsy specimens for the suspected pathology, very probably
derived from cells of the pathological tissues.
[0104] This screening is based in particular on the construction of
probes from blood cells and the hybridization of these probes on
filters bearing different markers specific to such and such a
pathology.
[0105] The invention describes the possibility of identifying
alterations in gene expression in blood cells, and this using
experimental models that mimic all or part of a human pathology
(eg., murine ALS model or hepatic tumor model).
The use of nucleic acid probes derived from blood cells of patients
with or without the screened pathology (neurodegenerative disease,
cancer, etc.) enables to screen for the existence of signatures
that are present in the experimental predictive banks created from
experimental disease models. The presence of common signatures
constitutes a diagnosis that the individual being tested is at risk
for developing such a pathology.
[0106] The invention also makes it possible to proceed in the
following manner: [0107] Blood samples from patients with or
without a disease being screened are pooled in order to constitute
a stock of RNA representing both healthy and pathological states.
[0108] This RNA is subjected to differential analyses according to
the methods set forth in the invention and cDNA banks
characteristic of pathological and healthy states are created.
[0109] These cDNA banks are then validated by hybridization with
probes prepared from individual blood samples from patients or
healthy subjects. [0110] The banks thus validated are then analyzed
through the use of probes prepared from blood samples from a large
population of subjects who consult a doctor for routine tests.
These banks constitute a diagnostic tool specific to the invention.
For example, a patient undergoing routine breast cancer screening
by mammography can have a small blood sample taken. A nucleic acid
probe prepared from such a sample then makes it possible to detect
very early signs indicative of development of a cancer, even in the
absence of positive radiographic findings.
[0111] The invention can be used in the form of a biochip. The
biochip makes use of the properties of hybridization in which two
so-called complementary strands of DNA bind to each other in a
highly specific manner The invention can also be used by
specifically seeking one or more DNA markers for the pathology by
using a DNA amplification method with oligonucleotide primers
specific for the desired DNA.
* * * * *