U.S. patent application number 14/792057 was filed with the patent office on 2016-01-07 for methods employing non-coding rna expression assays.
This patent application is currently assigned to SISTEMIC SCOTLAND LIMITED. The applicant listed for this patent is Jacinto ESTIBEIRO, John Gordon, Christopher Robert Hillier, Vincent O'Brien. Invention is credited to Jacinto ESTIBEIRO, John Gordon, Christopher Robert Hillier, Vincent O'Brien.
Application Number | 20160002722 14/792057 |
Document ID | / |
Family ID | 42078007 |
Filed Date | 2016-01-07 |
United States Patent
Application |
20160002722 |
Kind Code |
A1 |
ESTIBEIRO; Jacinto ; et
al. |
January 7, 2016 |
METHODS EMPLOYING NON-CODING RNA EXPRESSION ASSAYS
Abstract
There is disclosed a method comprising the steps of: carrying
out a plurality of expression assays, each expression assay
comprising the steps of: carrying out an intervention on a
biological system, measuring an expression profile of non-coding
RNAs in the biological system resulting from the intervention, and
storing an expression data set derived from the measured expression
profile, the said expression assays concerning either or both a
plurality of different interventions and a plurality of different
biological systems; and analysing the resulting expression data
sets to determine correlations between the effect on the expression
profile of non-coding RNAs of the respective intervention in groups
of two or more expression assays concerning either or both
different interventions or different biological systems.
Inventors: |
ESTIBEIRO; Jacinto;
(Gorebridge, GB) ; Gordon; John; (Tayport, GB)
; Hillier; Christopher Robert; (Glasgow, GB) ;
O'Brien; Vincent; (Ayr, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ESTIBEIRO; Jacinto
Gordon; John
Hillier; Christopher Robert
O'Brien; Vincent |
Gorebridge
Tayport
Glasgow
Ayr |
|
GB
GB
GB
GB |
|
|
Assignee: |
SISTEMIC SCOTLAND LIMITED
EDINBURGH
GB
|
Family ID: |
42078007 |
Appl. No.: |
14/792057 |
Filed: |
July 6, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13145208 |
Sep 7, 2011 |
9074241 |
|
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PCT/GB2010/000076 |
Jan 19, 2010 |
|
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14792057 |
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Current U.S.
Class: |
506/16 |
Current CPC
Class: |
C12Q 2600/142 20130101;
C12Q 1/6876 20130101; C12Q 2600/178 20130101; C12Q 2600/136
20130101; C12Q 1/6809 20130101; C12Q 1/6809 20130101; C12Q 2600/158
20130101; C12Q 2525/207 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 19, 2009 |
GB |
0900809.5 |
Oct 9, 2009 |
GB |
0917708.0 |
Claims
1.-26. (canceled)
27. An assay apparatus comprising miRNAs consisting of a reduced
group of miRNAs obtained by a method for identifying a group of
miRNAs for use in expression assays, the method comprising the
steps of: (1) carrying out a plurality of expression assays, each
expression assay comprising the steps of: carrying out at least one
intervention on a biological system comprising cultured cells or ex
vivo tissue, measuring an expression profile of a group of miRNAs
in the biological system resulting from the at least one
intervention, and storing an expression data set derived from the
measured expression profile, the said expression assays concerning
a plurality of different interventions; (2) analysing the resulting
expression data sets to determine correlations between the effect
on the expression profile of miRNAs of the at least one
intervention in groups of two or more expression assays concerning
the plurality of different interventions; (3) identifying a
sub-group of the group of miRNAs having expression profiles on
which the plurality of different interventions have a correlated
effect; and (4) selecting the sub-group of miRNAs for use in
further expression assays in which the expression profile of a
reduced group of miRNAs is measured, the reduced group including
only some of the group of miRNAs including at least the identified
sub-group of miRNAs, wherein the at least one intervention
comprises the application to the biological system of at least one
of a group comprising: ionizing radiation; continuously emitted or
pulsed electromagnetic radiation; acoustic energy; mechanical
intervention; the application of pressure, magnetic fields, or
electricity; changes in temperature; changes in osmolarity,
tonicity or pH of a growth medium; changes in fluid dynamics; and
mechanochemical signal transduction.
Description
FIELD OF THE INVENTION
[0001] The invention concerns methods employing non-coding RNA
expression assays. Embodiments of the invention addresses problems
including, but not limited to, determining similarities in the
mechanism by which two or more interventions affect biological
systems, identifying candidate therapeutic applications of test
agents and identifying new applications of therapeutic agents which
have previously been the subject of clinical trials in respect of
one or more indications.
BACKGROUND TO THE INVENTION
[0002] Issues concerning the invention will now be discussed with
reference to applications of microRNA (miRNA) expression assays,
however, the invention may employ expression assays concerning
other non-coding RNA molecules.
[0003] miRNAs are single-stranded RNA molecules having a length of
around 21 to 23 nucleotides. miRNAs were first described by Victor
Ambros in 1993 and since then over 2,000 papers on have been
published on the subject of miRNAs. There are predicted to be about
1,000 miRNAs in humans of which around 600 have been described and
experimentally validated to date, although some estimates place the
figure at tens of thousands. However, a recent report, which sought
to produce an expression atlas of miRNA in various human and rodent
tissues and cell lines, reported that around 300 miRNAs accounted
for 97% of all detected miRNAs.
[0004] miRNA is not translated into protein but instead regulates
the expression of one or more other genes. Known biology currently
shows that microRNAs target particular individual messenger RNAs
(mRNAs) or groups of mRNAs, thereby preventing their translation
or, less frequently, accelerating mRNA degradation. The mature
single stranded miRNA molecule complexes with the RNA-Induced
Silencing Complex (RISC) protein and binds to a partially
complementary sequence within the 3' untranslated region (3'-UTR)
of the protein coding mRNA from its target gene. Further proteins
are recruited to form a silencing complex and the expression of the
target gene product is repressed by a mechanism that blocks the
translation of the mRNA.
[0005] Although much remains to be discovered about the biology of
miRNAs and the composition and mechanism of the silencing complex
it is apparent that miRNAs are involved in the regulation of many
genes. MiRNAs are thought to regulate as many as 30% of all genes
(Xie et al, 2005) at the translational level. An miRNA can regulate
multiple genes and each gene can be regulated by multiple miRNAs.
Tissue-specific expression of miRNAs is thought to guide commitment
of cells to differentiate and/or actively maintain tissue identity.
This wide-ranging influence and interplay between different miRNAs
suggests that deregulated expression of a single miRNA or small
sub-set of miRNAs may result in complex disease traits (Lim et al,
2005, Nature). More than 50% of known human miRNAs reside in
genomic regions prone to alteration in cancer cells (Calin et al,
2004 PNAS, 101, 299-3004). Not surprisingly, the expression pattern
of miRNAs change in cancer and other disease states. This
information has begun to be used to classify and stage cancers,
reveal biomarkers for prognosis and response and provide a critical
determinant to guide therapeutic intervention, explain
chemosensitivity and inform the mechanisms of chemoresistance by
allowing the definition of specific miRNA expression patterns in
cancer stem cells.
[0006] Applications of miRNAs to research and the development of
possible new therapeutics have typically resulted from detailed and
time consuming analysis of the mechanisms by which miRNA expression
and processing is regulated and the mechanisms by which specific
miRNAs regulate mRNA translation. Specific drug targets have been
identified and research in connection with these drug targets in
ongoing. However, although thorough, this research paradigm is time
consuming and expensive.
[0007] Thus, the invention aims to provide alternative methods for
discovering practical applications of interventions, such as the
administration of a therapeutic agent, which do not require a
detailed understanding of the mechanism of action of the
intervention or the identification of a specific drug target. Some
embodiments of the invention address the problem of determining new
indications for known therapeutic entities or predicting
pharmacological properties of test agents, such as aspects of their
toxicological profile.
SUMMARY OF THE INVENTION
[0008] According to the present invention there is provided a
method comprising the steps of: [0009] (1) carrying out a plurality
of expression assays, each expression assay comprising the steps
of: carrying out an intervention on a biological system, measuring
an expression profile of non-coding RNAs in the biological system
resulting from the intervention, and storing an expression data set
derived from the measured expression profile, the said expression
assays concerning either or both a plurality of different
interventions and a plurality of different biological systems; and
[0010] (2) analysing the resulting expression data sets to
determine correlations between the effect on the expression profile
of non-coding RNAs of the respective intervention in groups of two
or more expression assays concerning either or both different
interventions or different biological systems.
[0011] By analysing expression data sets to determine similarities
between the effect of an intervention on the expression profile of
non-coding RNAs in groups of two or more expression assays, which
differ in terms of either or both the intervention which was
carried out and the biological system upon which the intervention
was carried out, correlations may be determined without it being
necessary to determine the mechanism by which one or more
interventions affect the expression profile of non-coding RNAs in
one or more biological systems.
[0012] Thus, where a second intervention is found to have an effect
on the expression profile of non-coding RNAs which is correlated
with the effect of a first intervention which is of known
therapeutic relevance, the second intervention can be treated as a
candidate for the same or a similar therapeutic application. There
is at least some possibility that the first and second
interventions will have the same, a similar, or a related mechanism
of action. This methodology is in direct contrast to known
strategies for discovering therapeutic interventions, in which a
specific target (such as a protein, nucleic acid or liquid
molecule) is identified, and analysed, the biology of the target is
studied in depth, and therapeutic interventions suitable to
modulate one or more activities of the target are developed by
rational and/or combinatorial methods.
[0013] One or more (and optionally all) said interventions may
comprise the application of one or more test agent to a biological
system, either simultaneously or sequentially. The one or more test
agent may be a chemical entity, for example, a molecule having a
molecular weight of less than 2,000 Daltons, less than 1,000
Daltons or less than 500 Daltons. The chemical entity may be
non-polymeric. The one or more test entity may be a biological
entity, for example, a biological macromolecule, such as a lipid,
an oligonucleotide, or a protein (e.g. an enzyme, an antibody, or
antibody fragment, humanized antibody or antibody fragment, phage
or ribosome displayed protein fragment, or a prion). The biological
entity may be a virus or bacteria. Thus, some or each of the
expression assays may measure the effect of a test agent on the
expression profile of non-coding RNAs in a biological system.
[0014] One or more said test agents may be a therapeutic agent. One
or more said test agents may be a therapeutic agent having a known
application to the treatment or prevention of a known condition.
One or more said test agents may be a therapeutic agent which has
been the subject of clinical trials (whether or not successfully)
in relation to one or more indications. However, one or more (and
optionally all) said interventions may comprise the application to
a biological system of one or more of a group comprising: ionising
radiation, continuously emitted or pulsed electromagnetic radiation
(for example, visible light, ultra-violet light, infra-red light),
acoustic energy (delivered through air or through a liquid medium),
mechanical intervention (for example, the application of pressure),
electricity, changes in temperature, changes in the osmolarity,
tonicity or pH of a growth medium, magnetic fields, changes in
fluid dynamics, and mechanochemical signal transduction. Thus, at
least some interventions may be interventions which are known to be
deleterious to the biological system. The expression profile
resulting from such deleterious interventions may be useful to
identify agents which reverse or prevent the deleterious
effects.
[0015] One or more said biological systems may comprise cells, such
as mammalian cells, for example, the cells of a human, a rabbit, or
a rodent (for example a mouse or a rat), or cultured insect,
amphibian or fish cell lines. One or more said biological systems
may comprise a mixture of cell types. The intervention is typically
carried out on cultured mammalian cells. The mammalian cells may be
stem cells or progenitor cells. By stem cells we refer to cells
which are capable of self-renewal and differentiation into at least
one other specialised cell type. However, one or more said
biological systems may be a whole organism, ex-vivo tissue, a
synthetic system or transformed cells. The one or more said
biological systems may be transgenic. Cultured cells may have
synchronous or asynchronous cell cycles. One or more said
interventions may be an intervention which changes the
differentiation or de-differentiation state of a stem cell or
progenitor cell, or which causes a stem cell or progenitor cell to
specialise, or to replicate while maintaining the characteristics
of a particular cell lineage or differentiation state.
[0016] The expression assays may be repeated and the expression
data sets which are analysed may be compiled from some or all
repeat experiments using equivalent interventions on equivalent
biological systems.
[0017] Correlations are typically between the expression of a
subset of the non-coding RNAs in connection with which expression
data is stored in the expression data sets. Correlations between
effects on the expression profile of non-coding RNAs are typically
correlations, which may be positive or negative, in the change in
the expression of one, or a small number of (e.g. two, three, five
or fewer than five, or ten or fewer then ten) non-coding RNAs
between two or more expression assays. A positive correlation may
comprise an increase in the expression of one or more non-coding
RNAs in each of two expression assays. A positive correlation may
comprise a decrease in the expression of one or more non-coding
RNAs in each of two expression assays. A negative correlation may
comprise an increase in the expression of one or more non-coding
RNAs in a first expression assay and a decrease in expression of
the same one or more non-coding RNAs in a second expression
assay.
[0018] In order to determine correlations between the effect on the
expression profile of non-coding RNAs of a respective intervention,
the method may further comprise measuring the expression profile of
non-coding RNAs in a suitable control assay, for example a control
assay in which the respective intervention is not carried out, or a
control assay comprising measuring the expression profile of
non-coding RNAs in a biological system prior to the respective
intervention being carried out. Differences between the expression
profile of non-coding RNAs in expression assays and corresponding
control assays may be determined. The stored expression data set
may be derived from a measured expression profile and an expression
profile of a corresponding control assay. The step of analysing the
resulting expression data may comprise taking into account
expression profiles from control assays. However, in some
applications it will not be necessary to carry out control assays.
For example, if a plurality of interventions are carried out on
equivalent biological systems it may be necessary only to analyse
data sets derived solely from the expression profiles resulting
from each expression assay to determine correlations between the
effect on the expression profile of non-coding RNAs of the
respective interventions.
[0019] It may be that at least some of the said correlations are
positive correlations, for example, similarities between the effect
on the expression of non-coding RNAs of the respective intervention
in groups of two or more expression assays. The step of analysing
the resulting expression data sets to determine correlations may
include the step of categorising (for example, clustering or
grouping) expression assays on the basis of similarities between
the expression data sets resulting from expression assays.
Advantageously, this may allow similarities in the mechanism by
which two or more different interventions have an effect directly
or indirectly on the expression profile of non-coding RNAs
(typically on the same or equivalent biological systems) to be
identified without a requirement for the nature of the shared
mechanism to be understood.
[0020] Thus, the method may be a method of determining that two or
more interventions have similar effects on the expression of one or
more non-coding RNAs. A first intervention may be the application
of a first therapeutic entity, having at least one known first
therapeutic application, and the method may be a method of
determining that there is a positive correlation between the effect
on non-coding RNA expression of a second intervention, comprising
the application of a second therapeutic entity. Accordingly, the
method may be a method of determining a possible new therapeutic
application (the first therapeutic application) for a known
therapeutic entity (the second therapeutic entity). The method may
further comprise the test of testing whether a second intervention
is applicable to the treatment of a said known first therapeutic
application.
[0021] The first intervention may be the application of an entity
without a known first therapeutic application, but which is known
to have pharmacological and toxicological profiles suitable for
deployment as a therapy. Thus, the method may be a method of
determining a possible new therapeutic application of a therapeutic
entity which has passed toxicology trials but failed to be found to
be efficacious, or more efficacious than a control therapeutic
entity, in clinical trials.
[0022] It may be that at least some of the said correlations are
negative correlations, for example, it may be determined that two
or more interventions have opposite effects on the expression of
one or more non-coding RNAs. Advantageously, a negative correlation
between the effect on the expression profile of non-coding RNAs of
a first intervention and a second intervention may indicate that
the second intervention could possibly be useful to reverse one or
more effects of the second intervention in therapy. Thus, the first
intervention may be an intervention which is known to have a
deleterious effect on the biological system, for example, the first
intervention may be the application of a toxin. In this case, the
method may comprise identifying the second intervention as a
candidate for the treatment or prevention of a condition known to
be caused by the first intervention.
[0023] Thus, a plurality of interventions may comprise the
administration of a toxin or a treatment which is deleterious to
the biological system. The method may therefore be part of a method
of determining candidate interventions (e.g. candidate therapeutic
entities) which may treat or prevent a condition known to be
causable by one or more other interventions. The method may be a
part of a method of determining candidates to treat or prevent side
effects of known therapeutic interventions (e.g. the application of
a therapeutic entity or a radiotherapy).
[0024] The method may be a method of predicting one or more aspects
of the toxicity of a test agent, for example, by detecting that the
expression profile of non-coding RNAs arising from a first
intervention is positively correlated with the expression profile
of an intervention which is known to have a deleterious effect on
the biological system, or positively correlated with the expression
profile of an intervention comprising the administration of an
agent, one or more aspects of the toxicology of which are
known.
[0025] The method maybe a method of determining that a first test
agent is a candidate agonist or antagonist of a second test agent,
or a specific target macromolecule, by determining a correlation
respectively between the effect on the expression profile of
non-coding RNA of the first test agent and the second test agent,
or a test agent which is a known agonist or antagonist of the
target molecule.
[0026] The method may comprise the step of grouping interventions
which have similar effects on the expression of non-coding RNAs.
The resulting expression profiles may be useful starting points for
further research to identify further therapeutic entities. The
method may be a method of determining changes in an expression
profile of non-coding RNAs associated with a group of
interventions, for example, a group of therapeutic entities. Thus,
the method may be a method of determining that a chemical or
biological entity has a mechanism of action on a biological system
which is related to the mechanism of action of another chemical or
biological entity on a biological system. Groups may be ordered in
a hierarchy.
[0027] Where an intervention is the application of a second test
agent to the biological system and the effect of the application of
the second test agent on the non-coding RNA expression profile is
found to be correlated (positively or negatively) to the effect of
another first test agent on the biological system, which first test
agent is known to be useful for the treatment or prevention of a
first condition, the second test agent, or test agents obtained by
modifying the second test agent, may be tested for efficacy in the
treatment or prevention of the first condition, or a condition
related to the first condition. Test agents which are found to be
efficacious for the treatment or prevention of the first condition,
or a condition related to the first condition, may be deployed from
the treatment or prevention of the relevant condition.
[0028] In some embodiments, expression assays are carried out in
which the same intervention, or group of interventions are carried
out on a plurality of different biological systems. Thus, the
method may enable the discovery of correlations between the effects
of interventions which are present in only some of the plurality of
different biological systems. In some embodiments, the plurality of
different biological systems are stem cells in different states of
differentiation or de-differentiation, for example, different
stages of development. Thus, the method may enable the discovery of
correlations between the effects of interventions on stem cells in
specific states of differentiation or de-differentiation. This
information is useful to investigate the mechanisms of development
and stem cell or progenitor cell differentiation and
de-differentiation. The plurality of different biological systems
may comprise mammalian cells in different disease states. An
intervention may be an intervention which causes stem cells or
progenitor cells to differentiate or de-differentiate, or drive the
attainment of a specific differentiation state or maintain the
stability of stem cells or progenitor cells in a particular
differentiation state.
[0029] The expression profile is related to the expression of at
least one, and typically a plurality of non-coding RNAs, preferably
at least 10, or more preferably at least 100 non-coding RNAs. The
expression profile may be related to the expression of one or more
transgenic non-coding RNAs functioning as markers. An expression
profile may include quantitative or qualitative measurements of the
level of expression of one or more non-coding RNAs. The level of
expression of one or more said non-coding RNA may be determined
indirectly via measurements of the amount or level of activation of
a reporter construct, for example, a transgenic reporter construct
incorporated into the genome of the biological system, or
maintenance episomally, in a particular biological system. The
expression profile is typically related to the amount of one or
more non-coding RNAs which are expressed in at least some
circumstances in the biological system, for example, the steady
state or peak amount of the one or more non-coding RNAs. However,
the expression profile may, for example, be related to the rate of
change of expression of one or more non-coding RNAs. In some
embodiments, the expression profiles are obtained using a
microarray.
[0030] The non-coding RNAs typically include microRNAs (miRNAs) and
may include either or both miRNA precursors and mature miRNAs. The
non-coding RNAs may comprise one or more of small interfering RNAs
(siRNA), piwi-interacting RNA (piRNA), small nuclear RNAs (snRNA),
and short hairpin RNA (shRNA). The non-coding RNAs may be
transgenic. Some or all of the RNAs may, for example, be transgenic
RNAs which function as reporters of non-coding RNA expression. The
non-coding RNAs may be episomal and the method may include the step
of introducing episomal DNA into the biological system, for example
by infection of a biological system with a virus, wherein the
episomal DNA can be transcribed to produce non-coding RNA which
constitute all or part of the profiled non-coding RNA.
[0031] Expression profiles may be measured for each non-coding RNA
in a group of non-coding RNAs and the method may comprise
identifying individual non-coding RNAs, or a sub-group of the group
of non-coding RNAs, which have expression profiles on which a
plurality of interventions have a correlated effect.
[0032] The plurality of interventions which have correlated effects
may be identified by the method of the invention, thus enabling
both interventions which have correlated effects on the expression
profile of a group of non-coding RNAs and the individual non-coding
RNAs or subgroup of non-coding RNAs within the group having
expression levels which are affected by the plurality of
interventions to be identified.
[0033] The plurality of interventions which have correlated effects
may be interventions which are previously known to have a related
mechanism of action, for example, the plurality of interventions
may comprise the administration of agents known or believed to have
the same or a similar mechanism of action, for example a class of
drugs. Thus, the invention provides a method of identifying the
individual non-coding RNAs or a subgroup of non-coding RNAs having
expression levels affected by the plurality of interventions.
[0034] The resulting identified individual non-coding RNAs or
identified sub-groups of non-coding RNAs may then be selected for
use in further expression assays in which the expression profile of
a reduced group of non-coding RNAs is measured, the reduced group
of non-coding RNAs including only some of the group of non-coding
RNAs, including, or optionally consisting of, at least the
identified individual non-coding RNAs or identified sub-groups of
non-coding RNAs. The effect of further interventions on the
expression profile of the reduced group of non-coding RNAs and
correlations between the effect of further interventions on the
expression profile of the reduced group of non-coding RNAs and the
effect of the said plurality of interventions on the expression
profile of the reduced group of non-coding RNAs can be thereby
determined. Thus, subsequent assays and tests may employ fewer
non-coding RNAs, reducing costs and increasing throughput. For
example, a reduced group of non-coding RNAs having expression
levels upon which a class of therapeutic agents have a correlated
effect may be used to screen candidate agents, either to find novel
therapeutically useful agents or to identify new indications for
known therapeutic agents.
[0035] The relevance of the expression level of a group or
sub-group of non-coding RNAs to discrimination between the effect
of biological interventions may be determined. The method may
comprise the step of ranking non-coding RNAs within the group or
sub-group depending on their relevance to discrimination between
the effects of biological interventions. The method may comprise
the step of ranking the effect on the expression of non-coding RNAs
in a group or sub-group of non-coding RNAs of a biological
intervention, or a group of biological interventions having a
correlated effect on the expression of non-coding RNAs. The
resulting rankings may be used to identify correlations between the
effects of biological interventions.
[0036] Correlations may be identified by statistical mathematical
methods, for example, principle component analysis. The effect of a
biological intervention on the expression of each of a plurality of
specific non-coding RNAs may be allocated one of a group of codes
indicative of properties of the effect of the biological
intervention on the expression of the respective non-coding RNA.
The resulting codes may be analysed to identify correlations.
[0037] The invention also extends to assay apparatus (for example a
test kit or a solid phase support having non-coding RNAs
immobilised thereto) having non-coding RNAs consisting of a said
reduced group of non-coding RNAs, obtained by the method of the
invention.
DESCRIPTION OF THE DRAWINGS
[0038] An example embodiment of the present invention will now be
illustrated with reference to the following Figure in which:
[0039] FIG. 1 is a flow diagram of a method according to the
invention;
[0040] FIG. 2 is a plot of the results from principal component
analysis for one biological intervention (a) and for one variable
(b);
[0041] FIG. 3 is a table giving statistical rankings of 11 miRNAs
by their p-value and q-value; and
[0042] FIG. 4 is a plot of data from principal component analysis
showing (a) a labelled sub-group of discriminatory miRNAs, and (b)
data from four intervention types showing how the expression data
from different intervention types cluster.
DETAILED DESCRIPTION OF AN EXAMPLE EMBODIMENT
[0043] In an example application of the invention, a database of
miRNA expression data sets (being an example of an expression data
set derived from a measured non-coding RNA expression profile) is
prepared. With reference to FIG. 1, suitable human cells are
cultured 2 by known methods and a test agent is administered 4 to
the cultured cells. A miRNA expression profile is then measured 6
using a sample of the treated cells, at one or more periods of time
after the intervention is made, to determine the expression level
of each of a number of miRNAs in the treated cells.
[0044] Two alternative methods for measuring the miRNA expression
profiles, microarray analysis and qualitative real-time PCR
analysis, are set out below.
(1) miRNA Microarray and Data Analysis
[0045] Total RNA from drug-treated (n=3) and control treated cells
(n=3) are isolated using a column-based kit from Exiqon A/S of
Vedbaek, Denmark. Two .mu.g of total RNA from each sample is
analysed by miRNA microarray. miRNA microarray analysis including
labelling, hybridization, scanning, normalization and data analysis
is commercially available from a number of sources, for example,
from Exiqon A/S. Briefly, RNA Quality Control is performed using
Bioanalyser 2100 microfluidics platform (Bioanalyser is a trade
mark of Agilent Technologies). Samples are labelled using the
Complete Labelling Hyb Kit from Agilent, following the provided
instructions.
(2) Quantitative Real-Time PCR
[0046] As with option (1) above, all cellular RNA is extracted
using a column-based kit from Exiqon and following the
manufacturer's instructions. Quantification of miRNAs by TaqMan
Real-Time PCR is carried out as described by the manufacturer
(Applied Biosystems of Foster City, Calif., USA). (TaqMan is a
trade mark of Roche Molecular Systems, Inc.). Briefly, 10 ng of RNA
is used as a template for reverse transcription (RT) using the
TaqMan MicroRNA Reverse Transcription Kit and miRNA-specific
stem-loop primers (Applied Biosystems). An aliquot (1.5 .mu.l) of
the RT product is introduced into 20 .mu.l PCR reactions which are
incubated in 96-well plates on the ABI 7900HT thermocycler (Applied
Biosystems) at 95.degree. C. for 10 min, followed by 40 cycles of
95.degree. C. for 15 s and 60.degree. C. for 1 min. Target gene
expression is normalized between different samples based on the
values of U48 RNA (a small, non-coding RNA) expression (or GAPDH,
if U48 is found to vary with drug treatment).
[0047] In each case, the resulting miRNA expression levels are
stored as 8 expression data sets. A large number of expression
assays are preferably carried out. Typically, many (e.g. hundreds
or thousands) of test agents are introduced to cell cultures and
analysed in this way to create a database of miRNA expression
data.
[0048] Once a suitably large database of miRNA expression data sets
are available, the expression data sets are analysed 10 to
determine correlations between the effects of each test agent on
miRNA expression and to create hierarchical clusters of test agents
which have similar effects on the miRNA expression profiles.
[0049] Methods for determining correlations between nucleic acid
expression data sets are well known to those skilled in the art.
For example, one method is to import microarray data obtained from
Exiqon A/S in the GPR format into a spreadsheet. (GPR is the data
format used by Genepix6 software, available from Molecular Devices
of Union City, Calif., USA. Genepix is a trade mark of Molecular
Devices). Spot intensities for each miRNA are analysed against
quality control and calibration spots provided on the miRNA array
(indicated by Genepix6 software as a negative flag). Values with
signal intensities below 50 are brought up to 0. For each of the
four replicate spots for each miRNA capture probe species, the
median value of the background corrected spot intensity is
calculated and imported into TMeV microarray analysis software
which performs hierarchical clustering and/or other statistical
analyses familiar to one skilled in the art. (TMeV is provided by
the Dana-Farber Cancer Institute, at the URL www.tm4.org).
[0050] Alternatively, GRP format expression data may be imported
into Genespring GX software, available from Agilent Technologies.
(Genespring GX is a trade mark of Agilent Technologies), normalised
to the 75.sup.th percentile and then processed using hierarchial
clustering and other statistical tools built into Genespring
GX.
[0051] Where positive correlations are found between the effects of
two or more test agents on the expression of one or more miRNAs,
this may be indicative that the test agents share the same, or a
related, mechanism of action. Thus, test agents which are found to
have similar effect on miRNA expression profile as an agent which
is known as a treatment for a condition can be identified 12 as
candidates for treatment of the same, or a related condition. This
may be useful to facilitate the repositioning of drugs which have
already been identified as potentially useful for one therapeutic
application. Candidate test agents can be tested to determine
whether they may be useful for treatment of the same, or a related
condition, or used as the starting point for further research. For
example, they might be modified using rational or combinatorial
design methodologies, a mimetic compound might be prepared and
tested and so forth. Candidate test agents can be tested 14 to
determine whether they are suitable for use as therapeutic entities
and, if, they are, deployed 16 as therapeutic entities.
[0052] It is especially useful to group test agents which have
similar effects on the expression of one or more miRNAs as this
classification by effect on miRNA expression may be reflected in a
similar or related mechanism of action, whether direct or indirect,
on miRNA expression levels.
[0053] Where negative correlations are identified, one test agent
might be identified as a candidate to prevent, mitigate or obviate
one or more undesirable affects of a further test agent or other
intervention. Thus, a test agent which is known to have an opposite
effect on the expression of one or more miRNAs to another test
agent which has an undesirable effect could be considered as a
candidate entity for the treatment or prevention of that
undesirable effect.
[0054] Advantageously, miRNA expression assays are carried out to
assess the effect of a range of interventions, including
interventions other than the administration of a chemical or
biological entity. For example, cells may be treated with
ultraviolet light, ionising radiation, acoustic waves and other
interventions which are deleterious to the cells. Where a test
agent can be identified which has an effect on the expression of
one or more miRNAs which is negatively correlated to the effect of
such interventions, the test agent may be a candidate for the
treatment or prevention of undesirable effects resulting from a
corresponding intervention in vivo. This may be useful to identify
agents for the prevention of damage caused by ultraviolet light or
as side effects from radiotherapies.
[0055] The method can be applied to the high-throughput screening
of large numbers of test agents (e.g. combinatorial libraries of
small chemical entities, peptides, peptidomimetics or polynucleic
acids). As new expression assays are carried out the resulting
expression data sets can be compared against previously stored
expression data sets to look for correlations between the effects
of screened test agents and agents which have been previously
assayed.
[0056] The method is typically best employed using a large database
of miRNA expression data sets. However, for some specific
applications it may only be necessary to have a small number of
miRNA expression data sets, or even one miRNA expression data set,
available for comparison with the miRNA expression data set
resulting from a new assay. This may be relevant in high-throughput
screens to find agents which have an effect on miRNA expression
which correlates positively or negatively with a particular
identified effect, for example, the effect of an agent which is
known or suspected as having a significant effect on miRNA
expression.
[0057] Thus, the invention is based on a principle that
similarities in mechanism of action, and therefore practical
applications, of test agents (such as chemical entities and
biologics) may be found through the comparative analysis of their
effects on the expression of miRNAs (and potentially other
non-coding RNAs) without it being essential to understand the
mechanism through which the test agents affect miRNA expression
profiles. This is in direct contrast to conventional drug discovery
and drug repositioning strategies in which a mechanism of action is
researched in depth to identify a drug target for use in screening
assays to discover agents which have a desired interaction with the
drug target.
Experimental Findings and their Implications
[0058] Using the methods described we have determined that it is
possible to determine potential modes of therapeutic application of
interventions based on the grouping of miRNA expression data.
Furthermore, the method can be employed to identify certain miRNAs,
having expression levels which are indicative of certain
therapeutic applications for interventions being screened. Such
indicative miRNAs will enable future intervention screening to
analyse a relatively small group of miRNA expression levels to
identify potential therapeutic applications of the interventions
being screened, and not the entire miRNA library.
[0059] An example of using a select small group of miRNAs to
determine potential therapeutic uses for an intervention is given
below.
[0060] During experiments described here, by way of a control, a
group of cells were treated with a drug solvent mix comprising
dimethyl sulphoxide, or DMSO, and phosphate buffered saline. It was
assumed that the drug solvent mix would not have an effect on miRNA
expression, and if it did, it would not be consistent with any of
the patterns associated with the drugs being tested. However, the
drug solvent mix was found to have a miRNA expression pattern
consistent with an HDAC inhibitor. Subsequently, it was found from
a literature review that DMSO had been shown to be an HDAC
inhibitor, confirming that unknown potential therapeutic properties
of drugs can be determined using the methods of the invention.
Materials and Methods
[0061] HeLa cells were cultured using standard methods. The cells
were split into DMEM medium.
[0062] The media was aspirated and the cell monolayer was washed
with an appropriate amount of Phosphate Buffered Saline (PBS, 8 g
NaCl, 0.2 g KCl, 1.44 g Na.sub.2HPO.sub.4 and 0.24 g
KH.sub.2PO.sub.4 dissolved in 800 ml of distilled H.sub.2O). The
PBS was aspirated.
[0063] The test agent in question was administered to the cells and
incubated for 48 hours.
RNA Extraction
[0064] RNA was isolated and purified from these cells using a
column-based kit from Exiqon the following procedure.
[0065] The medium the cells were grown on was aspirated and the
cell monolayer was washed with an appropriate amount of PBS. The
PBS was further aspirated.
[0066] 350 .mu.L of the lysis solution was added directly to a
culture plate. The cells were lysed by gently tapping the culture
dish and swirling buffer around the plate surface for five minutes.
The lysate was then transferred to a micro-centrifuge tube.
[0067] 200 .mu.L of 95-100% ethanol was added to the lysate and
mixed by vortexing for 10 seconds.
[0068] A column was assembled using one of the tubes provided in
the kit. 600 .mu.L of the lysate/ethanol was applied onto the
column and centrifuged for 1 minute at 14,000.times.g. The
flow-through was discarded and the spin column was reassembled with
its collection tube.
[0069] 400 .mu.L of the supplied wash solution was applied to the
column and centrifuged for 1 minute at 14,000.times.g. The
flow-through was discarded and the spin column was reassembled with
its collection tube.
[0070] The column was washed twice more by adding another 400 .mu.L
of wash solution and centrifuging for 1 minute at 14,000.times.g.
The flow-through was discarded and the spin column was reassembled
with its collection tube.
[0071] The column was spun for two minutes at 14,000.times.g to
thoroughly dry the resin and the collection tube was discarded.
[0072] The column was assembled into a 1.7 mL elution tube provided
with kit. 50 .mu.L of elution buffer was added to the column and
centrifuged for two minutes at 200.times.g followed by one minute
at 14,000.times.g.
[0073] The resulting purified RNA sample could be stored at
-20.degree. C. for a few days. For long-term storage of samples
were stored at -70.degree. C.
(1) miRNA Microarray and Data Analysis
Labelling
[0074] Purified RNA samples were labelled using a labelling kit
from Agilent.
[0075] The total RNA sample was diluted to 50 ng/.mu.L in
1.times.TE pH 7.5. 2 .mu.L of the diluted total RNA was added to a
1.5 mL micro-centrifuge tube and put on ice. Immediately prior to
use, 0.4 .mu.L 10.times. calf intestinal phosphatase buffer, 1.1
.mu.L nuclease free water and 0.5 .mu.L calf intestinal phosphatase
were gently mixed to prepare a calf intestinal alkaline phosphatase
master mix.
[0076] 2 .mu.L of the calf intestinal alkaline phosphatase master
mix was added to each sample tube for a total reaction volume 4
.mu.L, and was gently mixed by pipetting. The reaction volume was
incubated at 37.degree. C. in a circulating water bath for 30
minutes.
[0077] 2.8 .mu.L of 100% DMSO was added to each sample. Samples
were incubated at 100.degree. C. in a circulating water bath for
5-10 minutes and then immediately transferred to an ice bath.
[0078] 10.times.T4 RNA ligase buffer was warmed to 37.degree. C.
and spun until all precipitate had dissolved. Immediately prior to
use, 1 .mu.L of 10.times.T4 RNA ligase buffer, 3 .mu.L cyanine3-pCp
and 0.5 .mu.L T4 RNA ligase were gently mixed to make a ligation
master mix and put on ice.
[0079] 4.5 .mu.L of the ligation master mix was added to each
sample tube for a total reaction volume of 11.3 .mu.L. Samples were
gently mixed by pipetting and spun down. The samples were then
incubated at 16.degree. C. in a circulating waterbath for two
hours. The samples were then dried using a vacuum concentrator at
45-55.degree. C. and the samples were determined to be dry if, when
the tube was flicked the pellets did not move or spread.
Hybridization
[0080] 125 .mu.L of nuclease free water was added to the vial
containing lyophilised 10.times.GE blocking agent supplied with the
Agilent Kit and mixed.
[0081] The dried sample was resuspended in 18 .mu.L of nuclease
free water. 4.5 .mu.L of the 10.times.GE blocking agent was added
to each sample. 22.5 .mu.L of 2.times.Hi-RPM Hybridization buffer
was added to each sample and mixed well. The resulting samples were
incubated at 100.degree. C. for 5 minutes, and then immediately
transferred to an ice waterbath for a further 5 minutes.
[0082] A clean gasket slide was loaded into the Agilent SureHyb
chamber base ensuring the gasket slide was flush with the chamber
base. The hybridization sample was dispensed onto the gasket well
ensuring no bubbles were present.
[0083] An array was placed active side down onto the SureHyb gasket
slide and assembled with the SureHyb chamber cover to form an
assembled chamber. The assembled chamber was placed into a
hybridization oven set at 55.degree. C. and rotated at 20 rpm for
20 hours at that temperature.
[0084] The arrays were subsequently washed using the supplied GE
wash buffers before being scanned.
(2) Quantitative Real-Time PCR
Preparing the RT Reaction Master Mix
[0085] The components were thawed from frozen on ice. The RT
reaction master mix was prepared by mixing 0.15 .mu.L dNTPs (100
mM), 1 .mu.L MultiScribe Reverse Transcriptase (MultiScribe is a
trade mark of Applera Corporation) (50 U/.mu.L), 1.5 .mu.L
10.times. Reverse Transcription Buffer, 0.19 .mu.L RNase Inhibitor
(20 U/.mu.L), 4.16 .mu.L nuclease-free water, and then stored on
ice. Note that the volumes quoted above are per 15 .mu.L RT
reaction and were scaled up for the number of RT reactions to be
carried out.
Preparing the RT Reaction
[0086] For each 15 .mu.L RT reaction, 7 .mu.L RT master mix was
combined with 5 .mu.L total RNA. The RT Primers were thawed on ice
and 3 .mu.L of RT primer was added to the 12 .mu.L of the RT master
mix/total RNA in a 96-well plate well. The plate was kept on ice
until filled and then put into the thermal cycler.
Thermal Cycler Steps:
[0087] 16.degree. C. for 30 minutes [0088] 42.degree. C. for 30
minutes [0089] 85.degree. C. for 5 minutes [0090] 4.degree. C. for
as long as convenient
PCR Amplification
[0091] For each well, 10 .mu.L Taqman 2.times. Universal PCR Master
Mix was mixed with 7.67 .mu.L nuclease-free water, 1 .mu.L of
20.times. Taqman MicroRNA Assay mix and 1.33 .mu.L of the RT
product from the previous step. When all the wells were filled the
plate was sealed with an optical adhesive cover and centrifuged to
remove any air bubbles.
[0092] The plate was then loaded into a real-time capable thermal
cycler/PCR machine and the following program followed: [0093]
95.degree. C. for 10 minutes (Activation of the AmpliTaq Gold
Enzyme) [0094] 40.times.(95.degree. C. for 15 seconds, 60.degree.
C. for 60 seconds).
Data Analysis
[0095] Data from both of these techniques was normalised against
the spike-in miRNA spots for each plate, allowing data from
separate arrays to be compared.
[0096] Normalised data was analysed using Principal Component
Analysis, a standard technique well understood by those skilled in
the art to identify correlations between miRNA expression profiles,
and any grouping of data observed determined to be a consequence of
the action of the particular test agent applied to the original
cells on the expression of the individual miRNA.
[0097] FIG. 1 is a flow diagram of a method for obtaining an
expression profile for micro RNA.
[0098] FIG. 2 shows an example of an expression profile after
principal component analysis. Part (a) shows a three dimensional
projection of three principal components of the total
multidimensional expression data set of miRNA expression and
illustrates clustering of miRNA expression data for one treatment
type. Part (b) shows the data spread for the expression of single
miRNA exp
[0099] FIG. 3 shows a statistical ranking of 11 discriminatory
miRNAs labelled has-miR-1 through has-miR-11. The p-value is the
standard statistical test value of whether a result is
statistically significant or the result of chance (generally given
to be a p-value of .ltoreq.0.05) and the q-value being the p-value
corrected for multiple testing and provides a measure of the false
discovery rate. All p-values shown are much less than 0.05.
[0100] FIG. 4 (a) shows a projection of three principal components
of the multidimensional data set for miRNA expression for multiple
miRNAs and the clustering of miRNAs indicative of a potential
therapeutic applications. (b) shows a projection of three principal
components of the multidimensional data set for miRNA expression
for multiple miRNAs wherein the individual miRNAs are shaded to
indicate the therapeutic application for which the biological
intervention applied in their expression is used.
[0101] As can be seen, the results are clearly grouped and that
this grouping is according to the therapeutic use of the biological
intervention applied to the cells in which the miRNAs were
expressed. In other words, it is possible to determine that the
grouped biological interventions may have a similar mechanism of
action upon the cells to which they were applied, and the shared
mechanism resulted in similar effects on miRNAs expression
levels.
[0102] Biological interventions with similar mechanisms of action
may also have similar therapeutic properties and therefore they may
have similar therapeutic applications. Data presented in FIGS. 3
and 4 demonstrates that for the biological interventions tested,
the projection of three principal components of the
multidimensional data set for miRNA expression for biological
interventions of similar therapeutic application (for example,
anti-metabolites) did indeed group together, and that the groupings
of biological interventions with a different therapeutic use (for
example epigenetic modifiers) were grouped separately.
[0103] A database of miRNA expression patterns can be built up by
carrying out many biological interventions and analysing the
resulting changes in miRNA expression profile. Such a database
would enable identification of the therapeutic use, or potential
future therapeutic use, of an untested biological intervention by
comparing a miRNA expression profile of said untested biological
intervention with that in the database and determining whether the
said expression profile falls within one of the therapeutic
application groupings. If such a correlation occurs, the untested
biological intervention may be considered for that specific
therapeutic application.
[0104] Furthermore, building up a database of miRNA expression data
may reveal a subset of certain miRNAs that are indicative of a
certain therapeutic application. Once said subset of indicative
miRNAs are identified, future testing of new biological
interventions to find potential therapeutic applications, or
testing known biological interventions for new therapeutic
applications, can be carried out by looking at the expression
profiles of the subset of indicative miRNA expression profiles and
not the entire range of miRNAs produced by the cells.
[0105] The database of miRNA expression data may also be employed
to determine a subset of certain miRNAs, the expression levels of
which are most useful for discriminating between biological
interventions, or between groups of biological interventions known
or hypothesized to have similar modes of action. miRNAs may be
ranked in order of the relevance of their expression levels for
discriminating between biological interventions, or between groups
of biological interventions known or hypothesized to have similar
modes of action. miRNAs may be allocated a numerical value
indicative of the relevance of their expression levels for
discriminating between biological interventions, or between groups
of biological interventions known or hypothesized to have similar
modes of action. For example, the numerical value may be related to
the contribution of the expression level of an miRNA to the
variance of principle components.
[0106] As an alternative to, or in addition to, the comparison of
miRNA expression profiles using statistical methods such as
principal component analysis, the effect of a biological
intervention on the expression of each of a limited group of miRNAs
(for example, 10-50) may be identified and used to assign a code,
selected from a group of codes, to the effect of the biological
intervention on the expression of each respective miRNA. The
resulting codes may be compared to identify similarities in
effect.
[0107] For example, for each biological intervention (e.g. for each
screened compound) a 3-digit binary number may be allocated as a
code to each ranked miRNA based on:
1. If expression of the miRNA is unchanged (within normal limits of
experimental variability) in response to the biological
intervention, the first bit is set to 0. If expression has changed
significantly, the first bit is set to 1. 2. If a change in
expression level was identified and the change was an increase, the
second bit is set to 1. If the change resulting from the biological
intervention was a decrease, the second bit is set to 0. 3. If the
change in expression level was more than 4-fold, the third bit is
set to 1, otherwise it is set to 0.
[0108] Thus, the effect of a biological intervention level on the
expression of an miRNA is allocated a code having one of five
possible values:
1. No change in expression--000 2. Large increase in
expression--111 3. Small increase in expression--110 4. Large
decrease in expression--101 5. Small decrease in
expression--100
[0109] The effect of a biological intervention (for example, the
administration of a particular compound) on the expression level of
a group of miRNAs may be characterised by the associated code,
permitting identification of changes in expression level not
immediately apparent from principal component analysis, permitting
alternative methods of scoring the similarity of biological
interventions and rendering the resulting expression data
comprehensible by visual inspection.
[0110] Another way to characterise the effect of a biological
intervention and to determine correlations between the effects on
miRNA expression of different biological interventions is to carry
out an expression assay to determine the effects of a biological
intervention on the expression of each of a group (of typically 10
to 50) miRNAs and to rank the miRNAs in that group in order of the
effect, for example, in order from the miRNA in the group which has
the largest increase in expression to the miRNA in the group which
has the largest decrease in expression, or vice versa. The
resulting rankings are indicative of the effects of particular
biological interventions. Thus, the effect of other biological
interventions on the group of miRNAs may be measured and the miRNAs
in the group ranked in order of the effect. The resulting rankings
may be compared to enable correlations between the effect of
biological interventions to be identified.
[0111] A kit comprising plates operable to test the subset of
indicative miRNAs may be provided to significantly increase the
efficiency and speed with which biological interventions can be
screened for potential novel therapeutic applications.
[0112] Further variations and modifications may be made within the
scope of the invention herein disclosed.
REFERENCES
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motifs in human promoters and 3'-UTRs by comparison of several
mammals. Nature, 2005. 434(7031): p. 338-45 [0114] 2. Lim, L. P.,
et al., Microarray analysis shows that some microRNAs downregulate
large numbers of target mRNAs. Nature, 2005. 433(7072): p. 769-73
[0115] 3. Calin, G. A., et al., MicroRNA profiling reveals distinct
signatures in B cell chronic lymphocytic leukemias. Proc Natl Acad
Aci USA, 2004. 101 (32): p. 11755-60
* * * * *
References