U.S. patent application number 17/669675 was filed with the patent office on 2022-05-26 for functional prediction of cellular functions by means of microrna expresssion profiling in mesenchymal stem cells.
This patent application is currently assigned to SISTEMIC SCOTLAND LTD. The applicant listed for this patent is SISTEMIC SCOTLAND LTD. Invention is credited to Donald Dunbar, Elaine Gourlay, David Mallinson, Daria Olijnyk, James Reid.
Application Number | 20220160774 17/669675 |
Document ID | / |
Family ID | 1000006138172 |
Filed Date | 2022-05-26 |
United States Patent
Application |
20220160774 |
Kind Code |
A1 |
Mallinson; David ; et
al. |
May 26, 2022 |
FUNCTIONAL PREDICTION OF CELLULAR FUNCTIONS BY MEANS OF MICRORNA
EXPRESSSION PROFILING IN MESENCHYMAL STEM CELLS
Abstract
Non-coding RNA, such as miRNA, expression data derived from a
cell population is used to infer the propensity of that cell
population for a cellular functional effect for a pre-determined
purpose, which effect is temporally, procedurally or
interventionally separated from the cell population from which the
expression data is derived. Thereby, the cellular functional effect
of a cell population can be predicted in order to improve decisions
and selections to be made relating to the use of cells, e.g. from
cells deriving from different donors or batches, for use in
bioprocess application or cell therapeutics, in order to enhance
productivity, efficiency and/or efficacy.
Inventors: |
Mallinson; David; (Glasgow,
GB) ; Dunbar; Donald; (Glasgow, GB) ; Gourlay;
Elaine; (Glasgow, GB) ; Olijnyk; Daria;
(Glasgow, GB) ; Reid; James; (Glasgow,
GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SISTEMIC SCOTLAND LTD |
Glasgow |
|
GB |
|
|
Assignee: |
SISTEMIC SCOTLAND LTD
Glasgow
GB
|
Family ID: |
1000006138172 |
Appl. No.: |
17/669675 |
Filed: |
February 11, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
16300249 |
Nov 9, 2018 |
11266687 |
|
|
PCT/EP2017/061096 |
May 9, 2017 |
|
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17669675 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6883 20130101;
C12N 5/0663 20130101; A61P 35/00 20180101; A61K 35/17 20130101;
C12Q 2600/112 20130101; A61P 19/00 20180101; A61P 37/06 20180101;
C12Q 2600/158 20130101; C12N 5/0696 20130101; A61P 1/00 20180101;
C12Q 1/686 20130101; C12Q 2600/16 20130101; C12Q 2600/166 20130101;
A61P 9/00 20180101; C12Q 2600/178 20130101 |
International
Class: |
A61K 35/17 20060101
A61K035/17; C12Q 1/6883 20060101 C12Q001/6883; A61P 19/00 20060101
A61P019/00; A61P 9/00 20060101 A61P009/00; A61P 37/06 20060101
A61P037/06; A61P 1/00 20060101 A61P001/00; A61P 35/00 20060101
A61P035/00; C12N 5/0775 20060101 C12N005/0775; C12N 5/074 20060101
C12N005/074; C12Q 1/686 20060101 C12Q001/686 |
Foreign Application Data
Date |
Code |
Application Number |
May 13, 2016 |
GB |
1608497.2 |
May 9, 2017 |
GB |
1608081.4 |
May 9, 2017 |
GB |
1608086.3 |
Claims
1. A method of inferring propensity of a cell population for a
cellular functional effect for a pre-determined purpose, the method
comprising assaying the cell population against a pre-determined
non-coding RNA or panel of non-coding RNAs known or determined to
correlate with the cellular functional effect, generating
non-coding RNA expression data for the assayed non-coding RNA or
panel of non-coding RNAs and from the non-coding RNA expression
data identifying, determining or inferring a propensity for the
cellular functional effect, wherein the cellular functional effect
is a functional effect of a cell population which effect is
temporally, procedurally or interventionally separated from the
cell population from which the expression data or expression
profiles derive.
2. The method as claimed in claim 1, wherein the identifying,
determining or inferring a propensity for the cellular functional
effect is by comparing the expression data from the assay with
expression levels, trends or patterns known to be associated with
cellular functional effect.
3. The method as claimed in claim 1, which is for inferring a
cellular functional effect in a cell population by assaying that
cell population against the predetermined non-coding RNA or panel
of non-coding RNAs.
4. The method as claimed in claim 1, wherein the panel of
non-coding RNA comprises at least two non-coding RNA and preferably
up to six non-coding RNA.
5. The method as claimed in claim 4, wherein at least one
non-coding RNA is positively correlated and at least one non-coding
RNA is negatively correlated with the cellular functional
effect.
6. The method as claimed in claim 1, wherein the pre-determined
purpose is selected from a bioprocess applications, a cell therapy
application, patient stratification for cell therapy, cell growth
and donor selection.
7. The method as claimed in claim 1, wherein the cellular
functional effect is an effect on an applied cell population by a
tissue to which it is applied, an effect on a tissue to which a
cell population is applied, production of paracrine factors,
proliferation activity, differentiation tendency, engraftment
potential, immunosuppressive activity, migration potential,
response to activity inducing agents or response to assays.
8. The method as claimed in claim 1, which further comprises making
a selection or treatment decision based upon the identification,
determination or inference of propensity for the cellular
functional effect.
9. A method of treatment of a human or animal patient in need
thereof, the method comprising administering one or a plurality of
cell therapy doses to said patient, said cell therapy dose
effective in treating said patient by a cellular function effect as
between the cell therapy and the patient, the cellular function
effect having been inferred by use of non-coding RNA expression
profile, by measuring the non-coding RNA expression profile for the
cell therapy for a pre-determined non-coding RNA or panel of
non-coding RNAs known to correlate with the cellular functional
effect and inferring therefrom a relative propensity to the
cellular functional effect and determining therefrom with reference
to a predetermined dose for a pre-determined propensity of the one
or a plurality of cell therapy doses administered to said
patient.
10. The method as claimed in claim 9, wherein the treatment is one
of autologous cell therapy or allogenic cell therapy.
11. A cell population for use in the treatment of a condition in a
patient in need thereof, which treatment is mediated by a
pre-defined cellular functional effect, the cell population
provided in a dose or dosage regimen determined according to the
non-coding RNA expression of the cell population for a pre-defined
non-coding RNA or panel of non-coding RNAs that correlates with the
cellular functional effect to a degree that corresponds with one of
a plurality of possible doses or dosage regimen, by the method of
claim 9.
12. The cell population as claimed in claim 11 which are human
cells.
13. The cell population as claimed in claim 11 which cells are stem
cells or T-cells.
14. The cell population as claimed in claim 13, wherein the cells
are stem cells.
15. The cell population as claimed in claim 13, which cells are
stem cells selected from MSCs, induced pluripotent stem cells or
haematopoietic stem cells such as CD34+ cells.
16. The cell population as claimed in claim 15, wherein the stem
cells are MSCs.
17. The cell population as claimed claim 14, wherein the cellular
functional effect is differentiation into chondrocytes and the
pre-determined purpose is regenerative treatment of cartilage
tissue.
18. The cell population as claimed in claim 14, wherein the
cellular functional effect is differentiation into osteocytes and
the pre-determined purpose is regenerative treatment of bone
tissue.
19. The cell population as claimed in claim 14, wherein the
cellular functional effect is secretion of paracrine factors and
the pre-determined purpose is regenerative treatment of target
tissue.
20. The cell population as claimed in claim 14 wherein the cellular
functional effect is migration toward stromal cell-derived-factor 1
(SDF-1) and the pre-determined purpose is selection of donors of
stem cells for engraftment potential.
Description
RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
application Ser. No. 16/300,249, filed Nov. 9, 2018; that in turn
is a US National Phase of PCT Serial Number PCT/EP2017/061096,
filed May 9, 2017; that in turn claims prior to GB Application
Number 1608081.4, filed May 9, 2016; GB Application Number
1608086.3, filed May 9, 2016; and GB Application Number 1608497.2,
filed May 13, 2016; the contents of which are hereby incorporated
by reference.
FIELD OF THE INVENTION
[0002] This invention pertains generally to the predictability of
cellular function. More particularly, the invention relates to the
prediction functional outcome in the application of cell
populations to a purpose such as cell therapy.
BACKGROUND OF THE INVENTION
[0003] Many biological and clinical processes are facilitated by
the application of cells. Cell therapy (such as stem cell therapy)
in particular has clinical utility and is showing tremendous
promise for a range of indications by a range cell therapy
approaches.
[0004] Cell therapies (and other bioprocessing uses of cells)
suffer from variability in performance and predictability due to
the variability of phenotype and characteristics of both sources of
cells and target tissues (e.g. in prospective patients). This
results, potentially, in efficacy as well as cost and efficiency
being considerably affected and in some cases affects the viability
of the application.
[0005] It would be of enormous benefit to be able to predict
functional outcomes in order to make selection decisions--e.g.
therapeutic choices, donor selection or batch selection.
[0006] MicroRNAs (miRNA) are single-stranded RNA molecules having a
length of around 21 to 23 nucleotides which regulate gene
expression in cells at the translational level. Within a cell, a
single miRNA can regulate multiple genes and each gene can be
regulated by multiple miRNAs. There are currently 2588 known human
miRNAs. MiRNA are known to provide useful biomarkers for disease.
It is known that miRNAs can be used to characterize cells and can
be used to monitor when cells have changed (e.g. phenotypic
changes), or have differentiated, as discussed in
WO-A-2012/025709.
[0007] The present inventors have identified that microRNA (miRNA)
profile data can be used to predict a wide range of functional
outcomes in biological systems.
Problem to be Solved by the Invention
[0008] There is a need for improvements in the predictability of
functional outcome in cell applications in biological systems and
for making selection decisions.
[0009] It is an object of this invention to provide a method for
predicting the functional outcome the application of cells or
interventions on cells in a biological system.
[0010] It is a further object of this invention to provide an assay
for use in gathering data upon which to make a prediction of
functional outcome and/or make a selection of materials for use
according to the prediction of functional outcome.
[0011] It is a further object of this invention to provide a method
for selection of donor cells or cell batches based on a functional
outcome from the use of those cells.
SUMMARY OF THE INVENTION
[0012] In accordance with a first aspect of the invention, there is
provided a use of non-coding RNA (or miRNA) expression data or
expression profiles to identify, determine or infer propensity for,
or to predict, a cellular functional effect for a pre-determined
purpose.
[0013] In a second aspect of the invention, there is provided a
method of identifying, determining or inferring propensity for (or
predicting) a cellular functional effect for a pre-determined
purpose, the method comprising assaying against a pre-determined
non-coding RNA (or miRNA) or panel of non-coding RNAs (or miRNAs)
the expression of which is known or determined to correlate with
the cellular functional effect, generating non-coding RNA (or
miRNA) expression data for the assayed non-coding RNA (or miRNA) or
panel of non-coding RNAs (or miRNAs) and from the non-coding RNA
(or miRNA) expression data identifying, determining or inferring a
propensity for the cellular functional effect. For example, this is
typically by comparing the expression data from the assay with
expression levels (actual, e.g. ranges or thresholds, or relative),
trends or patterns known to be associated with cellular functional
effect.
[0014] In a third aspect of this invention, there is provided a
method of applying a cell population to a pre-determined purpose,
the method comprising inferring a cellular function effect for the
predetermined purpose by the method defined above and applying the
cell population for the pre-determined purpose.
[0015] In a fourth aspect of this invention, there is provided a
method for screening populations of donor-derived cells (e.g. stem
cells such as MSCs) for use in treatment of an indication or
condition or for further manipulation for later treatment of an
indication or conditions, the method comprising inferring a
cellular function effect pertinent to the treatment of the
indication or the further manipulation and in dependence of that
inference, selecting populations of donor-derived cells (or
selecting donors for further donation of cells) for use in the
treatment of the indication or the further manipulation.
[0016] In a fifth aspect of this invention, there is provided a
method for determining the dose of a population of cells (e.g. stem
cells, such as MSCs or T-cells) for administration to a patient for
treating a condition which depends upon a cellular functional
effect, the method comprising measuring a non-coding RNA (or miRNA)
expression profile for the cells for a pre-determined non-coding
RNA (or miRNA) or panel of non-coding RNAs (or miRNAs) known to
correlate with the cellular functional effect and inferring
therefrom a relative propensity to the cellular functional effect
and determining therefrom with reference, for example, to a
predetermined dose for a pre-determined propensity to the cellular
functional effect an actual dose to be administered to a
patient.
[0017] In a sixth aspect of this invention, there is provided a
method of treatment of a human or animal patient in need thereof,
the method comprising administering one or a plurality of cell
therapy doses to said patient, said cell therapy dose effective in
treating said patient by a cellular function effect as between the
cell therapy and the patient, the cellular function effect having
been inferred by use of non-coding RNA (or miRNA) expression
profile.
[0018] In a seventh aspect of this invention, there is provided a
population of cells selected to have a non-coding (or miRNA)
expression profile for a pre-defined non-coding RNA (or miRNA) or
panel of non-coding RNAs (or miRNAs) that correlates with a
pre-defined cellular functional effect optionally for a
pre-determined purpose.
[0019] In an eighth aspect of this invention, there is provided a
cell population for use in the treatment of a condition in a
patient in need thereof, which treatment is mediated by a
pre-defined cellular functional effect, the cell population
provided in a dose or dosage regimen determined according to the
non-coding RNA (or miRNA) expression of the cell population for a
pre-defined non-coding RNA (or miRNA) or panel of non-coding RNAs
(or miRNAs) that correlates with the cellular functional effect to
a degree that corresponds with one of a plurality of possible doses
or dosage regimen.
[0020] In a ninth aspect of the invention, there is provided a kit
for use to identify, determine or infer the propensity or relative
propensity of a population of cells for a cellular functional
effect, optionally for use in a pre-determine purpose, the kit
comprising primers for use in quantitative PCR (polymerase chain
reaction), primers being for a non-coding RNA (or miRNA) or panel
of non-coding RNAs (or miRNAs) known or identified as correlating
with the cellular functional effect.
[0021] In a tenth aspect of the invention, there is provided a
method of identifying or determining one or more non-coding RNA (or
miRNA) the expression of which in the cell population being assayed
is correlating with a cellular functional effect of the cell
population (e.g. when administered, subject to an intervention or
treated), preferably for a predetermined purpose, for use of said
one or more non-coding RNAs (or miRNAs) in a panel for identifying,
determining or inferring the cellular functional effect, the method
comprising: [0022] Sourcing cell populations intended for effecting
the cellular functional effect from multiple sources (e.g. multiple
donors or multiple production batches of different provenance);
[0023] Treating and using a first sample of each cell population to
isolate total RNA for non-coding (or miRNA) expression profiling
thereby generating an extensive non-coding RNA expression profile
data set for each cell population [or preferably a miRNA expression
profile data set for each cell population (e.g. based on at least
100 miRNAs, preferably at least 800, more preferably at least 1000
and most preferably at least 2000 miRNAs)]; [0024] Subjecting a
second sample of each cell population to an intervention designed
to elicit a cellular functional effect and the extent of the
cellular functional effect monitored by a known or conventional or
surrogate means to generate `response data`; [0025] Correlating the
extensive non-coding (or miRNA) expression profile data set with
response data for each cell population; [0026] Identifying
correlating non-coding RNA (or miRNA), preferably that correlate
positively or negatively with a correlation coefficient of at least
7 or at least 8; and [0027] selecting such correlating non-coding
RNA (or miRNA) as candidates for a miRNA expression panel for the
cellular functional effect.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1 is a correlation plot of miR-10b-5p expression levels
vs IDO-induction;
[0029] FIG. 2 is a correlation plot of miR-136-5p expression levels
vs IDO-induction;
[0030] FIG. 3 is a correlation plot of miR-140-3p expression levels
vs IDO-induction;
[0031] FIG. 4 is a correlation plot of miR-23a-3p expression levels
vs IDO-induction;
[0032] FIG. 5 is a correlation plot of miR-3651 expression levels
vs IDO-induction;
[0033] FIG. 6 is a correlation plot of miR-491-3p expression levels
vs IDO-induction;
[0034] FIG. 7 is a correlation plot of miR-574-3p expression levels
vs IDO-induction;
[0035] FIG. 8 is a correlation plot of miR-574-5p expression levels
vs IDO-induction;
[0036] FIG. 9 is a correlation plot of miR-1471 expression levels
vs percent migration for these miRNAs; and
[0037] FIG. 10 is a correlation plot of miR-1288-3p expression
levels vs percent migration for these miRNAs.
ADVANTAGES OF THE INVENTION
[0038] The use and method of the present invention enables decision
to be made and selections to be made relating to the use of cells,
e.g. from particular or categories of donors or batches, in
bioprocess applications or cell therapeutics that enhance
efficiency and/or efficacy. In particular, in cell therapy, the
method enables the decisions and selection in relation to cell
populations for the development of application of a cell therapy
for a patient with improved efficacy and efficiency.
DETAILED DESCRIPTION OF THE INVENTION
[0039] The invention concerns the use of non-coding expression data
or expression profiles to identify, determine or infer propensity
for a cellular functional effect, preferably for a pre-determined
purpose. The use can be effected, for example, by assaying against
a pre-determined non-coding RNA or panel of non-coding RNA, the
expression of which is already identified as correlated with the
cellular functional effect or a known surrogate or assay or other
process associated with the cellular functional effect. Preferably,
the panel of non-coding RNA comprises at least two non-coding RNA
and preferably up to six non-coding RNA.
[0040] The term `non-coding RNA` may include miRNA (microRNA) or
other non-coding RNA. The term `non-coding RNA` typically refers to
RNAs that do not encode a protein and generally encompass classes
of small regulatory RNAs. Other non-coding RNAs referred to above
may be, for example, small interfering RNA (siRNA),
piwi-interacting RNA (pi RNA), small nuclear RNA (snRNA), small
nucleolar RNA (snoRNA), extracellular RNA (exRNA), Small Cajal body
RNA (scaRNA) and short hairpin RNA (shRNA). Other non-coding RNAs
may further comprise transgenic non-coding RNAs which may function
as reporters of non-coding RNA expression. Other non-coding RNAs
may be episomal and the methods and/or uses described may require
initial steps in which episomal DNA is introduced into the cells
described herein whereupon the episomal DNA can be transcribed to
produce non-coding RNA which constitutes all or part of the
profiled non-coding RNA. In one embodiment, the term non-coding RNA
does not include non-coding RNAs known as teloRNA.
[0041] The term miRNA (microRNA) may include miRNA molecules and
either or both miRNA precursors and mature miRNAs as is apparent
from the context, but are preferably mature miRNAs.
[0042] Hereinafter, embodiments of the invention (and further
aspects) will be described by reference to miRNAs. Optionally as an
alternative to any or all of the embodiments of the invention
described hereinafter, the references to miRNA may instead be to
non-coding RNA or other non-coding RNA (such as those defied above)
where the context allows (e.g. other than when referring to
specific miRNAs).
[0043] Preferably, the invention is directed to the use of miRNA
expression data or expression profiles to identify, determine or
infer propensity for a cellular functional effect, preferably for a
pre-determined purpose. The use can be effected, for example, by
assaying against a pre-determined miRNA or panel of miRNA, the
expression of which is already identified as correlated with the
cellular functional effect or a known surrogate or assay or other
process associated with the cellular functional effect. Preferably,
the panel of miRNA comprises at least two miRNA and preferably up
to six miRNA.
[0044] The miRNA expression data or expression profile is
preferably expression data for a miRNA or panel of miRNAs known or
determined as having expression, expression levels or an expression
profile correlating with the cellular functional effect (or known
surrogate or assay or marker for or associated with the cellular
functional effect). The expression, expression level or expression
profile may be correlated positively or negatively with the
cellular functional effect.
[0045] The miRNA expression data may be expression data for at
least one miRNA, preferably at least two and more preferably up to
six miRNAs having a pre-determined correlating effect with the
cellular functional effect and preferably wherein at least one
miRNA is positively correlated and at least one miRNA is negatively
correlated with the cellular functional effect.
[0046] A cellular functional effect is any functional effect of a
cell or by a cell and is preferably associated with a functional
outcome. A cellular functional effect is typically an effect that
is potentially variable due, for example, to phenotypic differences
between cells or cell populations from different sources (e.g. in
different donors or patients) that may not readily determinable
until after the application or use or by carrying out an assay or
test for the effect or a surrogate marker. Preferably, the cellular
functional effect is a functional effect of a cell to be used for a
further or pre-determined use and preferably the cellular
functional effect is not demonstrable until after the cell has been
put to that further or pre-determined use. Given that with cell
therapies or other cell applications (such as bioprocess
applications), the timeframe and cost of expanding cell populations
or securing donations of cells for use in cell therapy (or
bioprocess development) is significant, it is a significant
impediment to cost effective therapy or use (e.g. in bioprocess
production) and effective further use (e.g. effective therapy for
the patient) to have variable or poor cellular functional effects
or functional outcomes. The use of miRNA expression data or
profiles and panels of miRNA in assays in accordance with the
present invention addresses the problems with variable or
unpredictable cellular functional effects and functional outcomes.
A functional outcome may be considered the result of the cellular
functional effect in the context of a pre-determined purpose.
[0047] A pre-determined purpose is preferably the purpose or use
that the cellular functional effect has or is associated with.
Typically, the effectiveness for the predetermined purpose is
dependent upon the cellular functional effect. The pre-determined
purpose may be any suitable purpose such as bioprocess
applications, regenerative medicine, cell therapy, patient
stratification (e.g. for cell or other therapy), cell growth or
donor selection for example.
[0048] By way of an example, a cellular functional effect may be
the migration potential of CD34+ cells toward SDF-1 or the IDO
induction activity of a population of MSCs, a functional outcome
may be respectively engraftment potential of CD34+ cells or
immunosuppressive activity of MSCs and a pre-determined purpose may
be respectively determination of dose for CD34+ for autologous
therapy or donor selection for, dose determination for or therapy
by T-cell suppression therapy (e.g. for Graft vs Host Disease).
[0049] The invention further comprises a method of identifying,
determining or inferring propensity for a cellular functional
effect for a pre-determined purpose, the method comprising
assaying, for example tissue or cells or cell populations, against
a pre-determined miRNA or panel of miRNAs known or determined to
correlate with the cellular functional effect, generating miRNA
expression data for the assayed miRNA or panel of miRNAs and
identifying, determining or inferring therefrom a propensity for
the cellular functional effect. This may be achieved by comparing
the expression data generated from the assay with expression levels
(actual, e.g. ranges or thresholds, or relative), trends or
patterns known to be associated (or correlated) with cellular
functional effect and optionally determining from the degree of
similarity or correlation with trends or patterns inferring a
relative or actual propensity. For a panel of miRNAs, the
expression data generated may be compared with a pattern for the
panel and a determination made according to a degree of correlation
with a pattern associated with or correlated with the cellular
functional effect. Preferably, a selection or decision may be made
to provide an enhanced functional outcome or enhanced efficacy of
the pre-determined purpose based on the determined propensity (or
relative propensity) for the cellular functional effect. That
selection may be, for example, the selection of cell populations
that meet or exceed a pre-determined threshold of propensity for
the cellular functional effect for use in the pre-determined
purpose or the selection of donors (as individuals or determined
donor groups determined or inferred to have a similar
characteristic) for donation of cells for a further use. That
selection may be, for example, the selection of those cell
populations that better demonstrate propensity for the cellular
functional effect (that is, have a better relative propensity) for
use in the pre-determined purpose.
[0050] Optionally, the expression levels or patterns (or degree of
correspondence or correlation) of the miRNA expression data may be
validated against the functional outcome of the cellular functional
effect in order to attribute an absolute value (e.g. a threshold)
or a scale to the miRNA expression data or correlation thereof with
the cellular functional effect or functional outcome from which a
precise prediction or inference can be derived and thereby a
decision or selection may optionally be made.
[0051] The invention further comprises a method of applying a cell
population to a pre-determined purpose, the method comprising
inferring (or determining) a cellular function effect for the
predetermined purpose by the method defined above and in dependence
of the inference (or determination) applying the cell population
for the pre-determined purpose.
[0052] The invention further comprises a method of identifying or
determining one or more miRNA correlating with a cellular
functional effect, preferably for a predetermined purpose, for use
in a panel of miRNA for inferring a cellular functional effect.
[0053] The invention further comprises the use of a panel of miRNA
(or miRNA adapted for assay form) in an assay for inferring a
cellular functional effect for which panel of miRNA the expression
has been determined as correlating with a cellular functional
effect.
[0054] The invention further comprises a kit for use in inferring a
cellular function effect for use in a pre-determined purpose. The
kit may comprise a panel of miRNAs (or equivalent reagents in a
form suitable for an assay) and optionally a protocol and method
for an assay and optionally a database or dataset relating to an
indication of expression levels (e.g. thresholds or ranges) or
patterns of miRNA associated with pre-defined and optionally
validated expression levels. The kit may typically be for use in
PCR (polymerase chain reaction), typically quantitative PCR and
comprise primers for a miRNA or panel of miRNAs.
[0055] In one embodiment, the pre-determined purpose is cell
therapy (for example stem cell therapy). According to this
embodiment, the invention further comprises a method of treatment
of a human or animal patient in need thereof, the method comprising
administering one or a plurality of cell therapy doses to said
patient, said cell therapy dose effective in treating said patient
by a cellular functional effect as between the cell therapy and the
patient, the cellular functional effect having been determined or
inferred by use of miRNA expression profile, preferably by assaying
against miRNAs known or determined to be correlated with the
cellular functional effect.
[0056] In another embodiment, the pre-determined purpose is a
bioprocess application, for example the production of a protein
(e.g. protein-based therapeutics such as monoclonal antibodies)
from cultures cells (e.g. murine myeloma cells, Chinese hamster
ovary cells, baby hamster kidney cells or human embryonic kidney
cells). The invention according to this embodiment is directed to
identifying, determining or inferring (or predicting) the
productivity of cells in producing the required molecules or
protein therapeutics and optionally selecting cells (e.g. cell
populations or cell batches, or cell lines or strains) for such
uses in dependence of their predicted productivity.
[0057] The term cell or cell population should be understood to
encompass any eukaryotic cell. For example a cell or cell
population may be or comprise a mammalian (adult, foetal or
embryonic) and preferably a human cell including, for example,
T-cells, progenitor cells (e.g. tissue-specific progenitor cells,
their intermediates stages differentiating to one or more terminal
states) or stem cells. The stem cells may be, for example, cell
populations comprising embryonic stem cells, induced pluripotent
stem cells, haematopoietic stem cells (e.g. CD34+ cells) or
mesenchymal stem/stromal cells.
[0058] The present invention in its broadest sense comprises at
least two general embodiments.
[0059] In a first general embodiment, it concerns use of miRNA
expression data of a cell, cell population or cell sample (together
referred to as cells hereafter) preferably for further use or for
use in a pre-determined purpose, to identify, determine or infer
propensity for a cellular functional effect associated with the
pre-determined purpose.
[0060] The use preferably is by assaying cells against a
pre-determined miRNA or panel of miRNA, the expression of which is
already identified as associated or correlated with the cellular
functional effect or a known surrogate or assay or other process
associated with the cellular functional effect. The panel of miRNA
may comprise any number of miRNA (e.g. one or more), the expression
of which are typically correlated with the cellular functional
effect (or another assay or process considered equivalent to the
cellular functional effect) and may be positively or negatively
correlated or may comprise one or more miRNA positively correlated
with the cellular functional effect and/or one or more miRNA
negatively correlated with the cellular functional effect.
Preferably, the panel of miRNA comprises at least two miRNAs and
preferably up to six miRNAs.
[0061] By expression data, it is meant expression levels, relative
expression levels or expression profiles or patterns of miRNA of
the cells.
[0062] The miRNA expression data is preferably expression data for
a miRNA or panel of miRNAs of the cells, the miRNA or panel of
miRNAs known or determined as having expression, expression levels
or an expression profile correlating with the cellular functional
effect (or known surrogate or assay or marker for or associated
with the cellular functional effect).
[0063] Preferably, the miRNA expression data is expression data for
two to six miRNAs having a pre-determined correlating effect with
the cellular functional effect and preferably wherein at least one
miRNA is positively correlated and at least one miRNA is negatively
correlated with the cellular functional effect.
[0064] The cellular functional effect according to this general
embodiment is a functional effect of a cell to be used for a
further or pre-determined use which cellular functional effect is
typically not readily demonstrable in the cells until after the
cell has been put to that further or pre-determined use or by some
other assay of the cells. Preferably, cellular functional effect
can be said to be a characteristic of the manner in which cells
interact at a cellular level in vivo or in vitro (according to the
pre-determined purpose).
[0065] A cellular functional effect is preferably a functional
effect of a cell or cell population or functional outcome of
application of the cell or cell population for a purpose, which
effect is separated temporally, procedurally or interventionally
from the cells or cell population from which the inference or
identification is being made (by means of assaying for miRNA
expression).
[0066] The cellular functional effect may be demonstrable a period
of time after the assay for inferring or identifying propensity for
the cellular functional effect, for example due to phenotypic
changes that the cell population may be pre-disposed to.
Alternatively, the cellular functional effect may be demonstrable
upon a process or procedure being applied to the cell population.
Alternatively the cellular functional effect may demonstrable after
an intervention performed on the cell population. Such an
intervention or procedural step may cause or induce a change in
phenotype or induce a particular effect. For example, the
intervention or procedural step may be an intervention to induce
expression or an assay to determine an effect or to induce an
effect. The intervention or procedural step may be a tagging step.
The intervention may be the in vitro culture of the cell
population, optionally under certain conditions, or serial passages
(e.g. 5, 10, 20, 40 or 80 passages) of a population of cells in in
vitro culture. The intervention or procedure step may comprise
administering the cell population to a patient (i.e. applying it to
an in vivo environment) optionally in combination with another
intervention. In broad terms, an intervention may be, for example,
any intervention which may be applied to or which acts on a cell
and which potentially may cause phenotypic changes or altered
function. Further examples of interventions may include the
application of one or more test agent to the cell population,
either simultaneously or sequentially, which 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, or non-polymeric, or a biological
entity (e.g. a biological macromolecule, such as a lipid, an
oligonucleotide, or a protein such as an enzyme, an antibody, or
antibody fragment, humanized antibody or antibody fragment, phage
or ribosome displayed protein fragment, or a prion, or a virus or
bacteria). Such a test agent intervention may be a therapeutic
agent. An intervention may comprise the application to a cell
population 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. An
intervention may be one that can potentially cause a change in the
differentiation or de-differentiation state of a stem cell or
progenitor cell, or which causes a stem cell or progenitor cell to
specialize, or to replicate while maintaining the characteristics
of a particular cell lineage or differentiation state.
[0067] Preferably, the cellular functional effect, the propensity
for which a population of cells is identified, determined or
inferred as having according to an aspect of the present invention,
in dependence upon non-coding or miRNA expression data or an
expression profile (the predictive data or predictive profile)
derived from the population of cells, is not contemporaneous with
the predictive data or predictive profile. Preferably, the cellular
functional effect is separated in time from the predictive data or
predictive profile, by which it is meant from time at which the
predictive data or predictive profile is derived from a population
of cells or sample thereof. The separation in time may be any
suitable time such as at least 15 minutes, at least an hour, at
least 24 hours, at least 1 week or at least 1 month. This
non-contemporaneous nature may be better defined in terms of cell
passages, e.g. separated by at least one passage, at least two
passages, at least 6 passages or at least 12 passages.
[0068] Optionally, according to an embodiment of the present
invention, the cellular functional effect is not demonstrable in
the cell population from which the non-coding RNA or miRNA
expression data or profile derives (at the time a sample is taken
for deriving such expression data or profile), but only after a
passage of time, application of a process or procedure, or of an
intervention on the cell population.
[0069] In a preferred embodiment of the use and method of the
invention, non-coding RNA or miRNA expression data or expression
profile may be derived from a population of cells (e.g. a first
population of cells) at a first occasion for use in identifying,
determining or inferring the propensity for a cellular functional
effect in a population of cells (e.g. a second population of cells)
at a second occasion. The second occasion and first occasion are
typically separated temporally, by process or by intervention on
the cell population. The second population of cells preferably
derives from the first population of cells by the passage of time,
application of a process or an intervention on the first population
of cell.
[0070] It is a preferred embodiment of the invention that the use
and method is for predicting a cellular functional effect (and
optionally a functional outcome) in a population of cells that is
to be subject to a passage of time, application of a process or
procedure or subject to an intervention and in particular to
predict the cellular functional effect (or identify, determine or
infer the propensity thereof) in a population of cells after a
passage of time, application of a process or procedure or after an
intervention, based upon non-coding RNA or miRNA expression data or
expression profiles derived from the population of cells before the
passage of time, the application of the process or procedure or
before the intervention.
[0071] The use according to this embodiment is preferably intended
to enable prediction, determination or inference as to propensity
of cells to a cellular functional effect leading to a functional
outcome for use in a pre-determined purpose. Preferably, this is in
order to make a selection (e.g. of cells from cell samples or
populations of nominally the same category or type but having
differing provenance, such as differing donor, differing storage
conditions or differing process or treatment conditions, to use
selected cells for a pre-determined purpose) or decision (e.g. to
proceed with a cellular therapy or not based on an absolute
determination of propensity or to determine quantity of cells to be
used for expansion or to determine doses for administration to a
cell therapy patient).
[0072] A pre-determined purpose according to this general
embodiment is a purpose or use of the cells that the cellular
functional effect has or is associated with. The pre-determined
purpose may be any purpose to which cells can be put which rely on
some functional effect of the cells (and a functional outcome of
applying the cells). The pre-determined purpose may be, for
example, a bioprocess application, a regenerative medicine
application, cell therapy, cell growth or donor selection.
[0073] The invention according to this general embodiment further
comprises a method of identifying, determining or inferring
propensity of cells for a cellular functional effect for a
pre-determined purpose, the method comprising assaying cells
against a pre-determined miRNA or panel of miRNAs known or
determined to correlate with the cellular functional effect,
generating miRNA expression data of the cells for the assayed miRNA
or panel of miRNAs and identifying, determining or inferring
therefrom the a propensity of the cells for the cellular functional
effect. This may be achieved by comparing the expression data
generated from the assay with expression levels (actual, e.g.
ranges or thresholds, or relative), trends or patterns known to be
associated (or correlated) with cellular functional effect and
optionally determining from the degree of similarity or correlation
with trends or patterns inferring a relative or actual propensity.
For a panel of miRNAs, the expression data generated may be
compared with a pattern for the panel and a determination made
according to a degree of correlation with a pattern associated with
or correlated with the cellular functional effect. Preferably, a
selection or decision may be made to provide an enhanced functional
outcome or enhanced efficacy of the pre-determined purpose based on
the determined propensity (or relative propensity) of the cells for
the cellular functional effect. That selection may be, for example,
the selection of cell populations that meet or exceed a
pre-determined threshold of propensity for the cellular functional
effect for use in the pre-determined purpose or the selection of
donors (as individuals or determined donor groups determined or
inferred to have a similar characteristic) for donation of cells
for a further use. That selection may be, for example, the
selection of those cell populations that better demonstrate
propensity for the cellular functional effect (that is, have a
better relative propensity) for use in the pre-determined
purpose.
[0074] Optionally, the expression levels or patterns (or degree of
correlation) of the miRNA expression data may be validated against
the functional outcome of the cellular functional effect in order
to attribute an absolute value (e.g. a threshold) or a scale to the
miRNA expression data or correlation thereof with the cellular
functional effect or functional outcome from which a precise
prediction or inference can be derived and thereby a decision or
selection may optionally be made.
[0075] The invention according to this general embodiment further
comprises a method of identifying or determining one or more miRNA
the expression of which in the cell population being assayed is
correlating with a cellular functional effect of the cell
population (e.g. when administered, subject to an intervention or
treated), preferably for a predetermined purpose, for use of said
one or more miRNAs in a panel for identifying, determining or
inferring the cellular functional effect. The method preferably
comprises: [0076] Sourcing cell populations intended for effecting
the cellular functional effect from multiple sources (e.g. multiple
donors or multiple production batches of different provenance);
[0077] Treating and using a first sample of each cell population to
isolate total RNA for miRNA expression profiling (e.g. using
Agilent miRNA microarrays: one version of which, Agilent MiRBase
version 16, contains 1199 human miRNAs; and another version of
which, Agilent MiRBase version 21, contains 2549 human miRNAs)
thereby generating an extensive (e.g. based on at least 100 miRNAs,
preferably at least 800, more preferably at least 1000 and most
preferably at least 2000 miRNAs) miRNA expression profile data set
for each cell population; [0078] Subjecting a second sample of each
cell population to an intervention designed to elicit a cellular
functional effect (said intervention being, for example,
administration to a patient with a target indication, treating with
an agent the response to which is to be predicted, subjecting to an
in vitro assay against something, subjecting to an inducing
environment such as to induce proliferation or to induce
differentiation into one or another differentiate, or expanding to
assess proliferation rate) and the extent of the cellular
functional effect monitored by a known or conventional or surrogate
means to generate `response data (e.g. therapeutic effect on
patient, degree of response to an agent, degree of induction, assay
results or proliferation rate); [0079] Correlating the extensive
miRNA expression profile data set with response data for each cell
population (e.g. using correlation methods as are known in the
art); [0080] Identifying correlating miRNA, preferably that
correlate positively or negatively with a correlation coefficient
of at least 7 or at least 8 (e.g. Pearson coefficient or other
accepted correlation measure); and [0081] selecting such
correlating miRNA as candidates for a miRNA expression panel for
the cellular functional effect.
[0082] The invention further comprises a method of applying cells
to a pre-determined purpose, the method comprising identifying,
determining or inferring a cellular function effect of the cells
for the predetermined purpose by the method defined above and in
dependence of the identification, determination or inference
applying the cells for the pre-determined purpose.
[0083] The invention according to this general embodiment further
comprises a kit for use in inferring a cellular function effect of
cells for use in a pre-determined purpose. The kit may comprise a
miRNA or a panel of miRNAs (or equivalent reagents in a form
suitable for an assay) and optionally a protocol and method for an
assay and optionally a database or dataset relating to an
indication of expression levels of miRNA associated with
pre-defined and optionally validated expression levels. The kit may
typically be for use in PCR (polymerase chain reaction), typically
quantitative PCR and comprise primers for a miRNA or panel of
miRNAs against which a cell population may be assayed.
[0084] The invention according to this embodiment further comprises
a cell population selected or adapted to have a propensity or
pre-determined degree of propensity for a cellular functional
effect, preferably for a pre-determined purpose. Optionally, the
degree of propensity may correspond to a correlation of at least
plus or minus 0.8.
[0085] The cellular functional effect may be any suitable cellular
functional effect which results in a functional outcome and is
ideally applicable to a purpose, such as culture, bioprocess or
therapy purposes. The cellular functional effect may be, for
example, a propensity for a cell (e.g. a stem cell such as a
mesenchymal stem/stromal cell) to differentiate to a particular
cell lineage, e.g. when subject to an induction and/or disposed in
a tissue type of that cell lineage. The cellular functional effect
may be, for example, the migration potential or the engraftment
potential of a cell (e.g. a stem cell) when administered in vivo.
The cellular functional effect may be, for example, the propensity
of a cell population to proliferate in culture and/or in vivo,
optionally when induced. The cellular functional effect may be, for
example, the propensity for the cells (e.g. mesenchymal stem cells)
to secrete factors, such as paracrine factors when subject to an
intervention such as particular culture conditions, an inducing
agent or being disposed in tissue of a particular type. The
cellular functional effect may, for example, the potential to be
affected by particular factors and enzymes, for example the
induction by interferon gamma of indoleamine-2,3-dioxygenase (IDO)
in mesenchymal stem cells.
[0086] In accordance with the invention, a miRNA or panel of miRNAs
may be used in uses or methods of the present invention applicable
to a particular cell type or category or range of types of cell
population.
[0087] In one embodiment, the cellular functional effect is the
proliferation character or the propensity for a cell population to
proliferate (or proliferation potential of a cell population). Such
proliferation character may be proliferation when in in vitro
culture, e.g. with inducement to proliferate. Preferably, the cells
are assayed for miRNA expression against a miRNA or panel of miRNAs
known to correlate with or be predictive of proliferation character
or potential. Preferably, the cells or cell populations are
mammalian cells, more preferably human cells. Preferably, the miRNA
expression data is derived from a panel of miRNA, which preferably
includes at least one miRNA positively correlated with
proliferation and/or at least one miRNA negatively correlated with
proliferation. Preferably, the panel of miRNA comprises at least
two miRNA and more preferably up to six miRNAs, one or more and
preferably all of which are selected from the aforementioned
miRNAs. The cells or cell populations according to this embodiment
may be any cell populations and preferably a mammalian cell
populations, e.g. for use in bioprocess applications such as murine
myeloma cells, Chinese hamster ovary cells, baby hamster kidney
cells or human embryonic kidney cells, and more preferably human
cell populations for various uses including in therapy, e.g.
T-cells or stem cells.
[0088] In another embodiment, the cellular functional effect is the
differentiation propensity or tendency to differentiate into one or
more cell lineages. According to this embodiment, there is provided
a miRNA or panel of miRNA for use according to the methods of the
present invention, for example to assay one or more cell
populations against for identifying, determining, inferring or
predicting the likelihood or tendency of the cells to differentiate
into a pre-determined or desired lineage tissue type (e.g. for
further study, for application as a therapeutic etc). The panel may
comprise candidates identified as defined above to indicate or
infer a propensity or preference for the cells to differentiate
into one or more of adipocytes, chondrocytes, osteocytes, myocytes
or one or more other lineages or sub-lineages common in various
tissue types, such as skin, heart tissue, vascular tissue, fibrous
extracellular tissue or nerve tissue. Preferably, the cells or cell
populations are mammalian cells, more preferably human cells. In
one preferred embodiment, a miRNA or panel of miRNAs are provided
that are indicative of the propensity of cells or cell populations
to differentiate into chondrocytes. Preferably, the miRNA
expression data is derived from a panel of miRNA, which preferably
includes at least one miRNA positively correlated with
chondrogenesis and/or at least one miRNA negatively correlated with
chondrogenesis. Preferably, the panel of miRNA comprises at least
two miRNA and more preferably up to six miRNAs. In another
preferred embodiment, a miRNA or panel of miRNAs are provided that
are indicative of the propensity of cells or cell populations to
differentiate into osteocytes. Preferably, the miRNA expression
data is derived from a panel of miRNA, which preferably includes at
least one miRNA positively correlated with osteogenesis and/or at
least one miRNA negatively correlated with osteogenesis.
Preferably, the panel of miRNA comprises at least two miRNA and
more preferably up to six miRNAs.
[0089] Further, according to this embodiment, there is provided a
kit for use to identify, determine or infer the propensity or
relative propensity of cell populations for differentiation into a
pre-defined or desired cell lineage such as adipocytes
(adipogenesis), chondrocytes (chondrogenesis) and osteocytes
(osteogenesis). A kit may typically be for use in quantitative PCR
(polymerase chain reaction), for example, and comprise primers for
a miRNA or panel of miRNAs known or identified as correlating with
differentiation into the pre-defined or desired cell lineage, such
as one of adipocytes (adipogenesis), chondrocytes (chondrogenesis)
and osteocytes (osteogenesis). The kit may further comprise
protocol and methods for the PCR assay. The kit may further
comprise a set of miRNA samples from cell populations, which miRNAs
are known or identified as correlating with differentiation into
the pre-defined or desired cell lineage, such as one of adipocytes
(adipogenesis), chondrocytes (chondrogenesis) and osteocytes
(osteogenesis) and correspond with the miRNA primers and/or
synthetic miRNA-specific oligonucleotides for the miRNA or panel of
miRNAs. The kit may further comprise a database or indication of
expression levels of miRNAs corresponding with a pre-defined
validated extent of propensity to differentiate into the
pre-defined or desired cell lineage such as one of adipocytes
(adipogenesis), chondrocytes (chondrogenesis) and osteocytes
(osteogenesis) and/or ranges of expression levels corresponding
with different extents of differentiation into one of adipocytes
(adipogenesis), chondrocytes (chondrogenesis) and osteocytes
(osteogenesis) and/or recommended dosage levels (e.g. for
regenerative cell therapy, e.g. in treating knee cartilage lesions)
corresponding to determined miRNA expression levels.
[0090] In one embodiment, the pre-determined purpose is a
bioprocess application. A bioprocess application may be a process
for producing proteins (e.g. protein-based therapeutics such as
monoclonal antibodies or enzymes). The invention may be used, for
example, to select cell populations, cell batches or cell strains
that may be productive for a particular purpose. The cells may be
any cells for use in bioproduction systems such as those mentioned
above for that purpose.
[0091] In one embodiment, the pre-determined purpose (or a further
purpose) is therapy or diagnosis. The therapy may be stem cell
therapy, such as regenerative therapy or immunomodulatory therapy.
The therapy may be immuno-oncology therapy. The therapy may be
heterologous, or homologous and may be allogenic or autologous.
[0092] In one embodiment, the pre-determined purpose is cell
therapy, for example stem cell therapy, for example regenerative
stem cell therapy. According to this embodiment, the invention
further comprises a method of treatment of a human or animal
patient in need thereof, the method comprising administering one or
a plurality of therapeutic doses of cells to said patient, said
cell therapy dose effective in treating said patient by a cellular
function effect of the cells as between the cell therapy and the
patient, the cellular function effect of the cells having been
determined or inferred by use of miRNA expression profile,
preferably by assaying the cells against miRNAs known or determined
to be correlated with the cellular functional effect.
[0093] The cell therapy may optionally be for autologous,
homologous or heterologous therapy, preferably homologous (e.g.
autologous or allogenic).
[0094] Optionally, the pre-determined purpose may be donor, batch
or cell population selection, for bioprocess or therapeutic
purposes. Cells from different sources (e.g. different donors) or
having different provenance (e.g. storage or culture conditions)
may have variable propensity to demonstrate a cellular functional
effect. For homologous (and heterologous) cell therapy, the
invention finds particular application in the selection of donor
cells or donor selection for cell therapy, especially for
particular indication having specific requirements of the cells.
Donor selection may be made according to any cellular functional
effect for a pre-determined or further purpose, such as a property
necessary for a bioprocess application or a therapeutic effect.
[0095] Optionally, the dose of cells to be administered may be
determined. For autologous cell therapy in particular (but also for
homologous and heterologous therapy), the invention finds
application in the determination of dose of cells to be
administered to a patient.
[0096] In one embodiment, the pre-determined purpose may be dose
determination of a cell population for use in a therapy. The dose
determination may be made based on the relative or actual
propensity of the cells for a cellular functional effect which is
associated with the efficacy or toxicity (or extent of side effect)
of the cell population for the therapy. For actual rather than
relative dose determination, it is preferred that the miRNA
expression of pre-determined miRNAs is correlated to a determinable
extent with a cellular functional effect associated with a
therapeutic effect (efficacy thereof) which extent is validated and
associated with one or more doses or ranges of doses.
[0097] The pre-determined purpose of the cells may be any further
use, which typically depends upon a cellular functional effect for
efficacy or for a desired level of efficiency. The pre-determined
purpose may be, for example, for regenerative medicine or any type
of cellular therapy on the human or animal body or cell culture or
any other application, such as bioprocess or research applications
for deriving new materials or the production of proteins or
metabolites.
[0098] The cells may be any suitable cells for which there is an
application associated with a cellular functional effect. The cells
may be eukaryotic or prokaryotic (e.g. bacterial cells) but are
preferably eukaryotic, more preferably mammalian cells.
[0099] Preferably the cells are human cells. Preferably the cells
are stem cells or T-cells.
[0100] The cells, especially stem cells, may be sourced from one or
multiple sources, which may be, for example, bone marrow, adipose
tissue, peripheral blood, skeletal muscle, endometrium, placenta,
umbilical cord blood, umbilical cord, Wharton's jelly, dental pulp
and cells derived from pluripotent cells.
[0101] Further, according to this embodiment, the invention
comprises cells for use in therapy by treatment or diagnosis, the
cells selected or adapted to have a miRNA expression profile that
have expression level (or patterns) commensurate or comparative
with or having a degree of correlation with a pre-determined miRNA
expression profile known or determined to be correlated with a
cellular functional effect associated with said treatment or
diagnosis.
[0102] Cell populations having a cellular functional effect
identified, determined or inferred by the methods of this invention
may be used for a variety of therapeutic applications and
indications. Therapeutic applications include, for example:
regenerative stem cell therapy, such as regeneration of target
tissue such as cartilage, bone, adipose tissue, skin, muscle, heart
tissue, vascular tissue, fibrous extracellular tissue, nerve
tissue, etc by administration of stem cells pre-disposed or
inducible to differentiate to lineages of such tissue or capable of
inducing growth of such tissue by secretion of paracrine factors or
by administration of in vitro induced differentiated
tissue-specific cells derived from stem cells, which regenerative
stem cell therapy may be useful in the treatment of tissue lesions,
tissue degenerative conditions (such as in autoimmune disorders
such as MS, Parkinson's disease or rheumatoid arthritis);
regenerative stem cell transplantation such as bone marrow
transplantation as part of a cancer therapy; immune-modulatory
therapy using stem cells pre-disposed or induced with an
immunomodulatory effect for use in the treatment of immune
disorders or responses, such as T-cell mediated immune disorders
(e.g. Graft versus Host Disease), and such as rheumatoid arthritis,
Crohn's disease and lupus; and targeted immuno-therapy such as
immuno-oncology by the allogenic or autologous administration of
T-cells pre-disposed or modified to target a tumour or target
cancer cells, such as chimeric antigen receptor modified
T-cells.
[0103] In one embodiment, the use and method is directed toward
T-cells, autologous or allogenic, optionally modified, for use in
therapy.
[0104] In one embodiment, the use and method is directed toward
stem cells. The stem cells may be obtained from one or a mixed
source of cells based upon expression of one or more cell surface
markers.
[0105] In one embodiment, the use and method is directed toward
mesenchymal stem cells for use in therapy.
[0106] In one embodiment, the use and method is directed toward
haematopoietic stem cells.
[0107] Such cells may be derived from bone marrow or peripheral
blood. Such cells may comprise cells that are CD34+ cells.
[0108] A cell population for use in treating a patient may be
characterized in that the cell population has a miRNA expression
profile consistent with or correlating with a pre-determined
expression profile for characterizing miRNAs for a particular
purpose.
[0109] There are now described more particular embodiments of the
invention falling within this general embodiment.
[0110] In one particular embodiment, the invention concerns the use
of miRNA expression data of mesenchymal stem cells (MSCs) from
donors or different donors (or categories of donors) to identify,
determine or infer the propensity or relative propensity of those
MSCs for indoleamine-2,3-dioxygenase (IDO) induction by
interferon-gamma (IFN-.gamma.). IDO induction is considered a
surrogate for the immune-suppressive potential of MSCs, thus the
present embodiment further provides the use of miRNA expression
data of MSCs from donors or different donors (or categories of
donors) to identify, determine or infer the propensity or relative
propensity of those MSCs for immunosuppressive action on T cells
(e.g. when administered to a patient, i.e. in vivo). Further, for
indications involving inflammation or T-cell proliferation, such as
Graft versus Host disease (e.g. in patients having received or
receiving transplanted tissue from a donor, such as stem cell or
bone marrow transplants), the invention according to this
particular embodiment preferably provides use of miRNA expression
data of mesenchymal stem cells (MSCs) from donors or different
donors (or categories of donors) to identify, determine or infer
the propensity or relative propensity of those MSCs for efficacy in
anti-inflammatory activity, in indications implicating T-cell
proliferation or in Graft versus Host disease. The same applies to
batches of MSCs rather than MSCs from a donor or different
donors.
[0111] Optionally, according to this particular embodiment, the
miRNA expression profile may be validated against the cellular
function or indication (e.g. Graft versus Host disease, e.g. in
terms of inhibition of T-cell proliferation) whereby the miRNA
expression profile levels may represent indicators of absolute
efficacy (rather than relative efficacy) and thus, dose may be
administered or selected accordingly.
[0112] Further, according to this particular embodiment, there is
provided a method of identifying, determining or inferring
propensity of MSCs from a donor or batch or different donors (or
categories of donors) or batches for indoleamine-2,3-dioxygenase
(IDO) induction by interferon-gamma (IFN-.gamma.) (or for efficacy
against or in the treatment of T-cell proliferation-mediated
indications or in the treatment of Graft versus Host disease) the
method comprising assaying cells against a pre-determined miRNA or
panel of miRNAs known or determined to correlate with IDO induction
by IFN-.gamma., generating miRNA expression data of the MSCs for
the assayed miRNA or panel of miRNAs and identifying, determining
or inferring therefrom the a propensity of the MSCs for the IDO
induction by IFN-.gamma. (or for efficacy against or in the
treatment of T-cell proliferation-mediated indications or in the
treatment of Graft versus Host disease). This may be achieved by
comparing the expression data generated from the assay with
expression levels (actual, e.g. ranges or thresholds, or relative),
trends or patterns known to be associated (or correlated) with IDO
induction of MSCs by IFN-.gamma. (or for efficacy against or in the
treatment of T-cell proliferation-mediated indications or in the
treatment of Graft versus Host disease) and optionally determining
from the degree of similarity or correlation with trends or
patterns inferring a relative or actual propensity. For a panel of
miRNAs, the expression data generated may be compared with a
pattern for the panel and a determination made according to a
degree of correlation with a pattern associated with or correlated
with the IDO induction of MSCs by IFN-.gamma. (or for efficacy
against or in the treatment of T-cell proliferation-mediated
indications or in the treatment of Graft versus Host disease).
Preferably, a selection or decision may be made to provide an
enhanced outcome or enhanced efficacy for the treatment of IDO
induction-mediated therapy (e.g. in the treatment of T-cell
proliferation-mediated indications or in the treatment of Graft
versus Host disease) based on the identified, determined or
inferred propensity (or relative propensity) of the MSCs for that
cellular functional effect. That selection may be, for example, the
selection of MSC populations that meet or exceed a pre-determined
threshold of propensity for IDO induction by IFN-.gamma. or the
selection of donors (as individuals or determined donor groups
determined or inferred to have a similar characteristic) for
donation of MSCs for use in therapy (thus a method of donor
selection is provided). That selection may be, for example, the
selection of those MSC populations that better demonstrate
propensity for IDO induction by IFN-.gamma. (that is, have a better
relative propensity) for use in therapy.
[0113] Optionally, the degree of correlation of the miRNA
expression data may be validated against the functional outcome of
the cellular functional effect in order to attribute an absolute
value or a scale to the miRNA expression data or correlation
thereof with the cellular functional effect or functional outcome
from which a precise prediction or inference can be derived and
thereby a decision or selection may optionally be made.
[0114] The invention according to this particular embodiment may
further comprise a method of administering MSCs for therapy (in
treatment of in the treatment of T-cell proliferation-mediated
indications or in the treatment of Graft versus Host disease), the
method comprising inferring (or determining) a propensity for IDO
induction by IFN-.gamma. in the MSCs by the method defined above
and in dependence of the inference (or determination) applying the
MSCs for the therapy.
[0115] The invention according to this particular embodiment
further comprises a method for the treatment of the human or animal
body by surgery, therapy or diagnosis by administering to a patient
in need thereof MSCs selected to have a propensity for IDO
induction. The invention according to this particular embodiment
comprises MSCs for use in surgery, therapy or diagnosis, said MSCs
selected to have a propensity for IDO induction.
[0116] Preferably the method and MSCs are for the treatment of
conditions treatable by MSC-mediated T-cell suppression (or
inflammatory or immune response disorders, typically involving
T-cell proliferation), such as Graft versus Host Disease.
[0117] The propensity may be a relative propensity or an absolute
propensity. Preferably, the MSCs having a propensity for IDO
induction are MSCs that have a miRNA expression profile for a
predetermined miRNA or panel of miRNAs known or identified to
correlate with IDO induction (or efficacy in MSC-mediated T-cell
suppression or in Graft versus Host disease), which said expression
profile meets pre-determined expression criteria consistent with
IDO induction potential. The pre-determined expression criteria may
be, for example, that the expression profile correlates with miRNA
expression in a standard or reference MSC that is known to be
susceptible IFN-7 induction of IDO to a reasonable, definable
extent, such correlation (e.g. Pearson correlation) being, for
example, at least plus or minus 0.7 more preferably at least plus
or minus 0.8, or that the expression levels of the miRNA or panel
of miRNAs meets particular expression levels or ranges (e.g. that
have been validated).
[0118] By expression data, it is meant expression levels, relative
expression levels or expression profiles or patterns of miRNA of
the cells.
[0119] Preferably, according to this particular embodiment, the
miRNA expression data (or profile) is miRNA expression data from a
miRNA or panel of miRNAs identified as having expression that
correlates with IDO induction by IFN-7. More preferably, the miRNA
or panel of miRNAs have expression that correlates with IDO
induction by IFN-7 with a correlation coefficient equal to or
greater than 7.0, more preferably equal to or greater than 8.0.
[0120] Preferably, the miRNA or panel of miRNAs comprise (and more
preferably consists) of miRNAs selected from one or more of
hsa-miR-10b-5p, hsa-miR-136-5p, hsa-miR-140-3p, hsa-miR-23a-3p,
hsa-miR-3651, hsa-miR-491-3p, hsa-miR-574-3p and hsa-miR-574-5p.
Preferably, the miRNA expression data is derived from a panel of
miRNA, which preferably includes at least one miRNA positively
correlated with IDO induction by IFN-7 (e.g. hsa-miR-10b-5p) and/or
at least one miRNA negatively correlated with IDO induction by
IFN-7 (e.g. hsa-miR-136-5p, hsa-miR-140-3p, hsa-miR-23a-3p,
hsa-miR-3651, hsa-miR-491-3p, hsa-miR-574-3p and hsa-miR-574-5p).
Preferably, the panel of miRNA comprises at least two miRNA and
more preferably up to six miRNAs.
[0121] Further, according to this particular embodiment, there is
provided a kit for use to identify, determine or infer the
propensity or relative propensity of MSCs from donors or different
donors or categories of donors IDO induction by interferon-gamma
(IFN-7) or for other consequential or corresponding purpose. A kit
may typically be for use in quantitative PCR (polymerase chain
reaction), for example, and comprise primers for a miRNA or panel
of miRNAs known or identified as correlating with IDO induction.
The kit may further comprise protocol and methods for the PCR
assay. The kit may further comprise a set of miRNA samples from
MSCs, which miRNAs are known or identified as correlating with IDO
induction and correspond with the miRNA primers and/or synthetic
miRNA-specific oligonucleotides for the miRNA or panel of miRNAs.
The kit may further comprise a database or indication of expression
levels of miRNAs corresponding with a pre-defined validated extent
of IDO induction and/or ranges of expression levels corresponding
with different extents of IDO induction and/or recommended dosage
levels (e.g. for cell therapy, e.g. in treating Graft versus Host
disease) corresponding to determined miRNA expression levels. The
miRNA or panel of miRNA are preferably as defined above.
[0122] Further, according to this particular embodiment, the miRNA
or panel of miRNAs may further comprise (or consist) or may in the
alternative comprise (or consist) of miRNAs selected from one or
more of hsa-miR-106a-5p, hsa-miR-106b-5p, hsa-miR-129-5p,
hsa-miR-137-3p, hsa-miR-145-5p, hsa-miR-151-5p, hsa-miR-17-5p,
hsa-miR-181a-5p, hsa-miR-186-5p, hsa-miR-206-3p, hsa-miR-20a-5p,
hsa-miR-214-3p, hsa-miR-300, hsa-miR-337-3p, hsa-miR-33a-5p,
hsa-miR-33b-5p, hsa-miR-367-3p, hsa-miR-520d-5p, hsa-miR-539-5p,
hsa-miR-543-3p, hsa-miR-580-3p, hsa-miR-675-3p, hsa-miR-720 and
hsa-miR-93-5p (which are optionally considered a non-redundant set
of predictive miRNAs). Preferably, the miRNA expression data is
derived from a panel of miRNA, which preferably includes at least
one non-redundant miRNA positively correlated with IDO induction by
IFN-7 (or surrogate predictive marker thereof) and/or at least one
non-redundant miRNA negatively correlated with IDO induction by
IFN-7 (or surrogate predictive marker thereof). Preferably, the
panel of miRNA comprises at least two miRNA and more preferably up
to six miRNAs.
[0123] Alternatively, the cells may be instead of T cells, B cells,
NK cells or DC cells, which offer a potential therapy autoimmune
disorders (including GVHD, but also Multiple Scleroses or Crohn's
disease).
[0124] In another particular embodiment, the invention concerns the
use of miRNA expression data of mesenchymal stem cells (MSCs) from
donors or different donors (or categories of donors) or from
batches or different batches to identify, determine or infer the
propensity or relative propensity of those MSCs for differentiation
into adipocytes (adipogenesis), chondrocytes (chondrogenesis) or
osteocytes (osteogenesis) and preferably to chondrocytes
(chondrogenesis) or osteocytes (osteogenesis). The propensity to
differentiate, e.g. into chondrocytes or osteocytes, has potential
direct implications on clinical applications of MSCs in
regenerative medicine, particularly in relation to the regeneration
of tissue, such as bone, cartilage, muscle, ligament, tendon, and
adipose tissues.
[0125] Further, for regenerative therapy indications, such as in
the regeneration of tissue, such as bone cartilage, muscle,
ligament, tendon and adipose tissue (e.g. in the regeneration of
knee cartilage, such as in the treatment of articular cartilage
lesions) the invention according to this particular embodiment
preferably provides use of miRNA expression data of mesenchymal
stem cells (MSCs) from donors or different donors (or categories of
donors) to identify, determine or infer the propensity or relative
propensity of those MSCs for efficacy in regenerative (and
preventative) effect, in such indications requiring tissue
regeneration. The same applies to batches of MSCs rather that MSCs
from a donor or different donors.
[0126] Optionally, according to this particular embodiment, the
miRNA expression profile may be validated against the cellular
function or indication (e.g. knee cartilage repair, e.g. in terms
of inhibition of propensity to differentiate into chondrocytes)
whereby the miRNA expression profile levels may represent
indicators of absolute efficacy (rather than relative efficacy) and
thus, dose may be administered or selected accordingly.
[0127] Further, according to this particular embodiment, there is
provided a method of inferring propensity of MSCs from a donor or
batch or different donors (or categories of donors) or batches to
identify, determine or infer the propensity or relative propensity
of those MSCs for differentiation into adipocytes (adipogenesis),
chondrocytes (chondrogenesis) and/or osteocytes (osteogenesis),
e.g. for use in regenerative therapy, the method comprising
assaying cells against a pre-determined miRNA or panel of miRNAs
known or determined to correlate with propensity to differentiate
into one of adipocytes (adipogenesis), chondrocytes
(chondrogenesis) and/or osteocytes (osteogenesis), generating miRNA
expression data of the MSCs for the assayed miRNA or panel of
miRNAs and identifying, determining or inferring therefrom the
propensity of the MSCs for differentiation into one of adipocytes
(adipogenesis), chondrocytes (chondrogenesis) and/or osteocytes
(osteogenesis) and thus propensity for efficacious regeneration of
adipose tissue, cartilage tissue or bone. This may be achieved by
comparing the expression data generated from the assay with
expression levels (actual, e.g. ranges or thresholds, or relative),
trends or patterns known to be associated (or correlated) with
propensity of the MSCs for differentiation into adipocytes
(adipogenesis), chondrocytes (chondrogenesis) and/or osteocytes
(osteogenesis) and optionally determining from the degree of
similarity or correlation with trends or patterns inferring a
relative or actual propensity. For a panel of miRNAs, the
expression data generated may be compared with a pattern for the
panel and a determination made according to a degree of correlation
with a pattern associated with or correlated with the propensity of
the MSCs for differentiation into adipocytes (adipogenesis),
chondrocytes (chondrogenesis) and/or osteocytes (osteogenesis).
Preferably, a selection or decision may be made to provide an
enhanced outcome or enhanced efficacy in regenerative therapy
(according to the tissue type being regenerated) based on the
identified, determined or inferred propensity (or relative
propensity) of the MSCs for that cellular functional effect. That
selection may be, for example, the selection of MSC populations
that meet or exceed a pre-determined threshold of propensity for
differentiation into one of adipocytes (adipogenesis), chondrocytes
(chondrogenesis) or osteocytes (osteogenesis) or the selection of
donors (as individuals or determined donor groups identified,
determined or inferred to have a similar characteristic) for
donation of MSCs for use in therapy (thus a method of donor
selection is provided). That selection may be, for example, the
selection of those MSC populations that better demonstrate
propensity for differentiation into adipocytes (adipogenesis),
chondrocytes (chondrogenesis) and/or osteocytes (osteogenesis)
(that is, have a better relative propensity) for use in
therapy.
[0128] Optionally, the degree of correlation of the miRNA
expression data may be validated against the functional outcome of
the cellular functional effect in order to attribute an absolute
value or a scale to the miRNA expression data or correlation
thereof with the cellular functional effect or functional outcome
from which a precise prediction or inference can be derived and
thereby a decision or selection may optionally be made.
[0129] The invention according to this particular embodiment may
further comprise a method of administering MSCs for regenerative
therapy (or selection for regenerative therapy, whether by direct
administration or deriving or inducing progenitor or differentiated
cells), the method comprising identifying, determining or inferring
a propensity for differentiation into one of adipocytes
(adipogenesis), chondrocytes (chondrogenesis) and/or osteocytes
(osteogenesis) by the method defined above and in dependence of the
identification, determination or inference applying the MSCs for
the therapy (or using the MSCs to derive cells such induced
differentiated cells or derived progenitor cells that may be used
in therapy).
[0130] The invention according to this particular embodiment
further comprises a method for the treatment of the human or animal
body by surgery, therapy or diagnosis by administering to a patient
in need thereof MSCs selected to have a propensity for
differentiation into one of adipocytes (adipogenesis), chondrocytes
(chondrogenesis) and osteocytes (osteogenesis), or differentiated
cells derived therefrom. The invention according to this particular
embodiment comprises MSCs (or differentiated cells derived from
MSCs) for use in surgery, therapy or diagnosis, said MSCs selected
to have a propensity for differentiation into one of adipocytes
(adipogenesis), chondrocytes (chondrogenesis) and osteocytes
(osteogenesis).
[0131] Preferably the method and MSCs are for the treatment of
conditions treatable by regenerative cell therapy (especially
regenerative stem cell therapy) such as MSC-mediated regenerative
therapy, such as cartilage repair or regeneration, adipose tissue
regeneration or bone repair or regeneration. For example, MSCs
pre-disposed or having a greater propensity to differentiate into
chondrocytes may provide enhanced efficacy in cartilage
regenerative therapy, such as in the repair of cartilage lesions,
whilst MSCs pre-disposed or having greater propensity to
differentiate into osteocytes may provide enhanced efficacy in
osteo-regenerative therapy, such as in the treatment of fractures
or osteoporosis.
[0132] The propensity may be a relative propensity or an absolute
propensity. Preferably, the MSCs having a propensity for
differentiation into adipocytes (adipogenesis), chondrocytes
(chondrogenesis) and/or osteocytes (osteogenesis) are MSCs that
have a miRNA expression profile for a predetermined miRNA or panel
of miRNAs known or identified to correlate with differentiation
into adipocytes (adipogenesis), chondrocytes (chondrogenesis)
and/or osteocytes (osteogenesis), which said expression profile
meets pre-determined expression criteria consistent with
differentiation into adipocytes (adipogenesis), chondrocytes
(chondrogenesis) and/or osteocytes (osteogenesis). The
pre-determined expression criteria may be, for example, that the
expression profile compares or correlates with miRNA expression in
a standard or reference MSC that is known to have a propensity or
relatively higher propensity for differentiation into adipocytes
(adipogenesis), chondrocytes (chondrogenesis) and/or osteocytes
(osteogenesis), e.g. to a reasonable, definable extent, such
correlation (e.g. Pearson correlation) being, for example, at least
plus or minus 0.7 more preferably at least plus or minus 0.8, or
that the expression levels of the miRNA or panel of miRNAs meets
particular expression levels or ranges (e.g. that have been
validated).
[0133] By expression data, it is meant expression levels, relative
expression levels or expression profiles or patterns of miRNA of
the cells.
[0134] Preferably, according to this particular embodiment, the
miRNA expression data (or profile) is miRNA expression data from a
miRNA or panel of miRNAs identified as having expression that
correlates with differentiation into respective one of adipocytes
(adipogenesis), chondrocytes (chondrogenesis) and osteocytes
(osteogenesis), more preferably that correlates with a correlation
coefficient equal to or greater than plus or minus 7.0, more
preferably equal to or greater than plus or minus 8.0.
[0135] Preferably, the miRNA expression data is derived from a
panel of miRNA, which preferably includes at least one miRNA
positively correlated with chondrogenesis and/or at least one miRNA
negatively correlated with chondrogenesis. Preferably, the panel of
miRNA comprises at least two miRNA and more preferably up to six
miRNAs.
[0136] Preferably, the miRNA expression data and miRNA or panel of
miRNAs for use in identifying, determining or inferring the
propensity or relative propensity of MSCs to differentiate into
osteocytes (osteogenesis) preferably includes at least one miRNA
positively correlated with osteogenesis and/or at least one miRNA
negatively correlated with osteogenesis. Preferably, the panel of
miRNA comprises at least two miRNA and more preferably up to six
miRNAs.
[0137] Further, according to this particular embodiment, there is
provided a kit for use to identify, determine or infer the
propensity or relative propensity of MSCs from donors or different
donors or categories of donors for differentiation into one of
adipocytes (adipogenesis), chondrocytes (chondrogenesis) and
osteocytes (osteogenesis). A kit may typically be for use in
quantitative PCR (polymerase chain reaction), for example, and
comprise primers for a miRNA or panel of miRNAs known or identified
as correlating with differentiation into one of adipocytes
(adipogenesis), chondrocytes (chondrogenesis) and osteocytes
(osteogenesis). The kit may further comprise protocol and methods
for the PCR assay. The kit may further comprise a set of miRNA
samples from MSCs, which miRNAs are known or identified as
correlating with differentiation into one of adipocytes
(adipogenesis), chondrocytes (chondrogenesis) and osteocytes
(osteogenesis) and correspond with the miRNA primers and/or
synthetic miRNA-specific oligonucleotides for the miRNA or panel of
miRNAs. The kit may further comprise a database or indication of
expression levels of miRNAs corresponding with a pre-defined
validated extent of propensity to differentiate into one of
adipocytes (adipogenesis), chondrocytes (chondrogenesis) and
osteocytes (osteogenesis) and/or ranges of expression levels
corresponding with different extents of differentiation into one of
adipocytes (adipogenesis), chondrocytes (chondrogenesis) and
osteocytes (osteogenesis) and/or recommended dosage levels (e.g.
for regenerative cell therapy, e.g. in treating knee cartilage
lesions) corresponding to determined miRNA expression levels. The
miRNA or panel of miRNA are preferably as defined above for each
purpose.
[0138] In another particular embodiment, the invention concerns the
use of miRNA expression data of CD34+ cell populations to identify,
determine or infer the propensity or relative propensity of those
CD34+ cells to migrate toward Stromal cell-derived-factor 1
(SDF-1).
[0139] CD34+ cell populations are typically derived from blood or
bone marrow and are used in autologous and allogenic therapy in a
number of indications. To exert the desired therapeutic effect,
appropriate dosing is necessary and this tends to be based upon
cell count, an approach that has questionable legitimacy since the
characteristics of the cells in any CD34+ cell population is
variable and difficult to characterize. In particular, CD34+ cell
populations can have highly variable numbers of haematopoietic stem
and progenitor cells. Engraftment potential is considered to be
related to the therapeutic efficacy of CD34+ cells and, associated
therewith, SDF-1 migration potential is considered to correspond to
the potential of such transplanted cells to migrate to specific
tissues or sites of injury leading to engraftment.
[0140] It is believed that the in vivo SDF-1 migration potential of
CD34+ cell populations is related to the therapeutic efficacy of
CD34+. An SDF-1 migration assay has been used to show that SDF-1
migration potential corresponds with rate of engraftment after
autologous cell transplantation (see:) and such an assay has been
suggested as an alternative approach to assessing engraftment
potential and dosing. However, the assay is convoluted and time
consuming and incompatible with the time pressures of autologous
CD34+ cell therapy.
[0141] This particular embodiment is directed toward a rapid screen
that is a surrogate for an in vitro SDF-1 migration assay. The
propensity or relative propensity of those CD34+ cells to migrate
toward SDF-1 is determined from the propensity or relative
propensity of those CD34+ cells to migrate toward SDF-1 according
to an in vitro SDF-1 migration assay. The use is directed toward
the engraftment of stem cells in autologous stem cell therapy.
[0142] Indications for autologous or allogenic CD34+ haematopoietic
stem cell therapy include haematopoietic reconstitution in the
treatment of cancers (such as leukaemias), metastatic breast
cancer, neuroblastoma, cardiovascular diseases such as myocardial
ischemia (e.g. to increase exercise capacity), myocardial
infarction and ischemic stroke, multiple sclerosis and autoimmune
disorders. The application of this particular embodiment has
particular utility in autologous therapy.
[0143] The use according to this particular embodiment is
preferably directed toward determining or inferring graft potential
of CD34+ cell populations and thus would be useful in determining a
dose for autologous CD34+ haematopoietic stem cell therapy.
[0144] Optionally, according to this particular embodiment, the
miRNA expression profile may be validated against the SDF-1
migration assay whereby the miRNA expression profile levels may
represent indicators of absolute graft potential and thus a dose
may be administered or selected accordingly.
[0145] Further, according to this particular embodiment, there is
provided a method of identifying, determining or inferring
propensity of CD34+ cell populations for SDF-1 migration potential
for use in CD34+ stem cell therapy, especially autologous therapy,
the method comprising assaying cells against a pre-determined miRNA
or panel of miRNAs known or determined to correlate with SDF-1
migration potential (typically by way of correlating with SDF-1
migration in an in vitro assay), generating miRNA expression data
of the CD34+ cell population for the assayed miRNA or panel of
miRNAs and identifying, determining or inferring therefrom the
propensity of the of CD34+ cell populations for SDF-1 migration
potential (or engraftment potential) for use in CD34+ stem cell
therapy. This may be achieved by comparing the expression data
generated from the assay with expression levels (actual, e.g.
ranges or thresholds, or relative), trends or patterns known to be
associated (or correlated) with propensity of the of CD34+ cell
populations for SDF-1 migration potential and optionally
determining from the degree of similarity or correlation with
trends or patterns inferring a relative or actual propensity. For a
panel of miRNAs, the expression data generated may be compared with
a pattern for the panel and a determination made according to a
degree of correlation with a pattern associated with or correlated
with the propensity of the of CD34+ cell populations for SDF-1
migration potential.
[0146] Preferably, a determination as to the dose of CD34+ cells to
be used in therapy may be made to enhance efficacy of the CD34+
stem cell therapy based on the determined propensity of the CD34+
cells for that cellular functional effect. That determination as to
dose may be based upon, for example, comparison (e.g. expression
levels or patterns) or correlation of the miRNA expression data for
a population of the CD34+ cells to be administered with levels or
pattern of expression of the miRNA or panel of miRNAs known to
correlate with and validated against the cellular functional effect
(that is, SDF-1 migration potential as determined by in vitro
assay), or even functional outcome in vivo. Thus, one may attribute
an absolute value or a scale to the miRNA expression data or
correlation thereof with the cellular functional effect or
functional outcome from which a precise prediction or inference can
be derived and thereby a determination as to dose may be made.
[0147] The invention according to this particular embodiment may
further comprise a method of administering CD34+ cell populations
for therapy (in autologous stem cell therapy, for example), the
method comprising identifying, determining or inferring a
propensity for SDF-1 migration potential of the CD34+ cell
population by the method defined above and in dependence of the
identification, determination or inference applying the CD34+ cells
for the therapy.
[0148] The invention according to this particular embodiment
further comprises a method for the treatment of the human or animal
body by surgery, therapy or diagnosis by administering to a patient
in need thereof a CD34+ cell dose selected to have a propensity for
SDF-1 migration. The invention according to this particular
embodiment comprises CD34+ cells for use in surgery, therapy or
diagnosis, said CD34+ cell populations selected to have a
propensity for SDF-1 migration.
[0149] Preferably the method and CD34+ cell populations are for the
treatment of conditions treatable by CD34+ haematopoietic stem
cell-mediated therapy, such as one or more of those indications
mentioned above in connection with this particular embodiment.
[0150] The propensity may be a relative propensity or an absolute
propensity. Preferably, the CD34+ cell populations having a
propensity for SDF-1 migration are CD34+ cell populations that have
a miRNA expression profile for a predetermined miRNA or panel of
miRNAs known or identified to correlate with SDF-1 migration, which
said expression profile meets pre-determined expression criteria
consistent with SDF-1 migration potential. The pre-determined
expression criteria may be, for example, that the expression
profile correlates with miRNA expression in a standard or reference
CD34+ cell population that is known to have a high propensity (or
defined or desirable propensity) for SDF-1 migration to a
reasonable, definable extent, such correlation (e.g. Pearson
correlation) being, for example, at least plus or minus 0.7 more
preferably at least plus or minus 0.8, or that the expression
levels of the miRNA or panel of miRNAs meets particular expression
levels or ranges (e.g. that have been validated).
[0151] By expression data, it is meant expression levels, relative
expression levels or expression profiles or patterns of miRNA of
the cells.
[0152] Preferably, according to this particular embodiment, the
miRNA expression data (or profile) is miRNA expression data from a
miRNA or panel of miRNAs identified as having expression that
correlates with SDF-1 migration potential. More preferably, the
miRNA or panel of miRNAs have expression that correlates with SDF-1
migration potential with a correlation coefficient equal to or
greater than 7.0, more preferably equal to or greater than 8.0.
[0153] Preferably, the miRNA or panel of miRNAs comprise (and more
preferably consists) of miRNAs selected from one or both of
has-miR-1471 and has-miR-1288-3p. Both these miRNAs have a positive
correlation with SDF-1 migration potential.
[0154] Further, according to this particular embodiment, there is
provided a kit for use to identify, determine or infer the
propensity or relative propensity of CD34+ cell populations to
SDF-1 migration potential or for other consequential or
corresponding purpose. A kit may typically be for use in
quantitative PCR (polymerase chain reaction), for example, and
comprise primers for a miRNA or panel of miRNAs known or identified
as correlating with SDF-1 migration potential. The kit may further
comprise protocol and methods for the PCR assay. The kit may
further comprise a set of miRNA samples from CD34+ cell
populations, which miRNAs are known or identified as correlating
with SDF-1 migration potential and correspond with the miRNA
primers and/or synthetic miRNA-specific oligonucleotides for the
miRNA or panel of miRNAs. The kit may further comprise a database
or indication of expression levels of miRNAs corresponding with a
pre-defined validated extent of SDF-1 migration and/or ranges of
expression levels corresponding with different extents of SDF-1
migration and/or recommended dosage levels (e.g. for cell therapy)
corresponding to determined miRNA expression levels. The miRNA or
panel of miRNA are preferably as defined above.
[0155] In another particular embodiment, the invention concerns the
use of miRNA expression data of T-cells from a donor (or modified
T-cells) to identify, determine or infer the propensity or relative
propensity of those T-cells for proliferation.
[0156] Immunotherapies based upon cell-based adoptive transfer of
T-cells are showing great promise and very effective results in
treatment of certain conditions, notably oncology indications.
Typically, autologous therapies involve donor T-cells from a
patient that are optionally subject to a modification (e.g. to
surface antigens to target a tumour) and are tumour reactive.
Allogenic therapies are also under development and in clinical
trials. These donor T-cells or modified donor T-cells are expanded
in vitro before administration to a patient in therapy. A critical
constraint and technical challenge for such therapy is the ability
to generate therapeutically suitable numbers of cells. It would be
advantageous at an early stage of a process to be able to screen a
donor or a donor's T-cells for proliferation potential or
proliferation rate in the development of a therapy. This particular
embodiment provides a screen for proliferation potential of donor
T-cells which may be used in immunotherapies, especially immuno
oncology therapies, such as cell-based adoptive transfer of
T-cells.
[0157] T-cell populations (or modified T-cell populations) for use
in such therapeutic applications require to be expanded to provide
therapeutic doses for patients.
[0158] Indications for autologous or allogenic T-cell based
immunotherapies, such as by cell-based adoptive transfer of
T-cells, include autoimmune disorders and cancers such as solid
tumors, cervical cancer, lymphoma, leukemias, bile duct cancer,
neuroblastoma, lung cancer, breast cancer, sarcoma, melanoma,
CD19-expressing haematologic malignancies and CD19+ B cell
malignancies, including B-cell acute lymphoblastic leukaemia (which
may harbor rearrangement of the mixed lineage leukaemia), and may
be mediated by, for example, chimeric antigen receptor modified
T-cells.
[0159] The use according to this particular embodiment is
preferably directed toward identifying, determining or inferring
proliferation potential of T-cell or modified T-cell populations
and thus would be useful in determining the suitability of a donor
for development of a T-cell based therapeutic or in determining the
quantity of donor T-cell material required to expand to a
therapeutic dose in a pre-determined time.
[0160] Optionally, according to this particular embodiment, the
miRNA expression profile may be validated against proliferation
rate whereby the miRNA expression profile levels may represent
indicators of absolute proliferation potential and thus a decision
or selection may be made accordingly.
[0161] Further, according to this particular embodiment, there is
provided a method of identifying, determining or inferring
propensity of T-cell or modified T-cell populations for
proliferation potential for use in cell-based adoptive transfer
T-cell therapy, especially autologous therapy, the method
comprising assaying cells against a pre-determined miRNA or panel
of miRNAs known or determined to correlate with T-cell
proliferation potential, generating miRNA expression data of the
T-cell population for the assayed miRNA or panel of miRNAs and
identifying, determining or inferring therefrom the a propensity of
the T-cell population for proliferation potential. This may be
achieved by comparing the expression data generated from the assay
with expression levels (actual, e.g. ranges or thresholds, or
relative), trends or patterns known to be associated (or
correlated) with proliferation and optionally determining from the
degree of similarity or correlation with trends or patterns
inferring a relative or actual propensity. For a panel of miRNAs,
the expression data generated may be compared with a pattern for
the panel and a determination made according to a degree of
correlation with a pattern associated with or correlated with
proliferation potential or rates. Preferably, a determination as to
the quantity of T-cells required from the donor, or the suitability
of the donor for developing a therapy, may be made. That
determination may be based upon, for example, a comparison (or
degree of correlation) of the miRNA expression data for the miRNA
or panel of miRNA the expression levels or patterns of which are
validated against the cellular functional effect (that is,
proliferation potential), in order to attribute an absolute value
or a scale to the miRNA expression data or correlation thereof with
the cellular functional effect or functional outcome from which a
precise prediction or inference can be derived and thereby a
determination as to dose may be made.
[0162] The invention according to this particular embodiment may
further comprise a method of administering T-cell or modified
T-cell populations for therapy (in autologous or allogenic
cell-based adoptive transfer T-cell therapy, for example), the
method comprising inferring (or determining) a propensity for
proliferation potential of the donor T-cell or modified T-cell
population by the method defined above and in dependence of the
inference (or determination), modifying and expanding the T-cells
to a therapeutic dose and applying the T-cell dose for the
therapy.
[0163] The invention according to this particular embodiment
further comprises a method for the treatment of the human or animal
body by surgery, therapy or diagnosis by administering to a patient
in need thereof a T-cell or modified T-cell dose selected to have a
propensity for proliferation. The invention according to this
particular embodiment comprises T-cell populations or modified
T-cell populations for use in surgery, therapy or diagnosis, said
T-cell populations selected to have a high propensity for
proliferation.
[0164] Preferably the method and T-cell populations are for the
treatment of conditions treatable by cell-based adoptive transfer
of T-cells, such as by CAR T-cell therapy.
[0165] The propensity may be a relative propensity or an absolute
propensity. Preferably, the T-cell populations having a propensity
for proliferation are T-cell populations that have a miRNA
expression profile for a predetermined miRNA or panel of miRNAs
known or identified to correlate with a high proliferation
potential, which said expression profile meets pre-determined
expression criteria consistent with high proliferation potential.
The pre-determined expression criteria may be, for example, that
the expression profile compares or correlates with miRNA expression
in a standard or reference T-cell population that is known to have
a high propensity (or defined or desirable propensity) for
proliferation to a reasonable, definable extent, such correlation
(e.g. Pearson correlation) being, for example, at least plus or
minus 0.7 more preferably at least plus or minus 0.8, or that the
expression levels of the miRNA or panel of miRNAs meets particular
expression levels or ranges (e.g. that have been validated).
[0166] By expression data, it is meant expression levels, relative
expression levels or expression profiles or patterns of miRNA of
the cells.
[0167] Preferably, according to this particular embodiment, the
miRNA expression data (or profile) is miRNA expression data from a
miRNA or panel of miRNAs identified as having expression that
correlates with high proliferation potential. More preferably, the
miRNA or panel of miRNAs have expression that correlates with high
proliferation potential with a correlation coefficient equal to or
greater than 7.0, more preferably equal to or greater than 8.0.
[0168] Preferably, the miRNA expression data is derived from a
panel of miRNA, which preferably includes at least one miRNA
positively correlated with proliferation and/or at least one miRNA
negatively correlated with proliferation. Preferably, the panel of
miRNA comprises at least two miRNA and more preferably up to six
miRNAs.
[0169] Further, according to this particular embodiment, there is
provided a kit for use to identify, determine or infer the
propensity or relative propensity of T-cell populations to
proliferation potential or for other consequential or corresponding
purpose. A kit may typically be for use in quantitative PCR
(polymerase chain reaction), for example, and comprise primers for
a miRNA or panel of miRNAs known or identified as correlating with
high (or desirable) proliferation potential. The kit may further
comprise protocol and methods for the PCR assay. The kit may
further comprise a set of miRNA samples from T-cell populations,
which miRNAs are known or identified as correlating with a
proliferation potential and correspond with the miRNA primers
and/or synthetic miRNA-specific oligonucleotides for the miRNA or
panel of miRNAs. The kit may further comprise a database or
indication of expression levels of miRNAs corresponding with a
pre-defined validated extent of proliferation and/or ranges of
expression levels corresponding with different extents of
proliferation recommended donor quantities for expansion
corresponding to determined miRNA expression levels. The miRNA or
panel of miRNA are preferably as defined above.
[0170] Whilst the invention defined herein refers to identifying,
determining or inferring a propensity for a cellular functional
effect, it may likewise be defined as identifying, determining or
inferring whether or not there will be (or is likely to be) a
cellular functional effect or a sufficient or desirable level of a
cellular functional effect. In this general embodiment, this
directed in particular toward a cell or cell population, it may be
used to predict functional outcomes and make determinations and
selection or take action determined therefrom.
[0171] In a second general embodiment, it concerns use of miRNA
expression data derived from target tissue or target cells (e.g. a
prospective patient) to which a cell population is to be applied
(e.g. for treatment by a pre-determined cell therapy) to determine
the propensity for that cell population (e.g. cell therapy) to have
a particular effect on the target tissue or target cells (in the
prospective patient).
[0172] Target tissue is intended to include various tissue types
within which cells relevant to, for example, a therapeutic
treatment may exist or move to. Target tissue includes biofluids,
such as blood plasma. Target tissue includes tissue to which
treatment effects are to be applied, but include other tissues
where indications or predictions of treatment by miRNA expression
profiles may be determinable.
[0173] Thus, the miRNA expression data can be used to stratify a
patient population for receiving the cell therapy.
[0174] The use preferably is by assaying cells or tissue from a
patient (or multiple patient) against a pre-determined miRNA or
panel of miRNA, the expression of which is already identified as
associated or correlated with the cellular functional effect of a
cell or a known surrogate or assay or other process associated with
the cellular functional effect. The panel of miRNA may comprise any
number of miRNA, the expression of which are typically correlated
with the cellular functional effect (or another assay or process
considered equivalent to the cellular functional effect) and may be
positively or negatively correlated or may comprise one or more
miRNA positively correlated with the cellular functional effect
and/or one or more miRNA negatively correlated with the cellular
functional effect. Preferably, the panel of miRNA comprises at
least two miRNA and preferably up to six miRNA.
[0175] By expression data, it is meant expression levels, relative
expression levels or expression profiles or patterns of miRNA of
the cells.
[0176] The miRNA expression data is preferably expression data for
a miRNA or panel of miRNAs of the cells, the miRNA or panel of
miRNAs known or determined as having expression, expression levels
or an expression profile correlating with the cellular functional
effect (or known surrogate or assay or marker for or associated
with the cellular functional effect).
[0177] Preferably, the miRNA expression data is expression data for
two to six miRNAs having a pre-determined correlating effect with
the cellular functional effect and preferably wherein at least one
miRNA is positively correlated and at least one miRNA is negatively
correlated with the cellular functional effect.
[0178] The cellular functional effect according to this general
embodiment is a functional effect of a cell to be used for a
further or pre-determined use which cellular functional effect is
typically not readily demonstrable in the cells until after the
cell has been put to that further or pre-determined use or by some
other assay of the cells. Preferably, cellular functional effect
can be said to be a characteristic of the manner in which cells
interact at a cellular level in vivo or in vitro (according to the
pre-determined purpose).
[0179] A cellular functional effect is preferably a functional
effect of a cell or cell population or functional outcome of
application of the cell or cell population for a purpose, which
effect is separated temporally, procedurally or interventionally
from the cells or cell population from which the inference or
identification is being made (by means of assaying for miRNA
expression).
[0180] Preferably, the cellular functional effect, the propensity
for which a population of cells is identified, determined or
inferred as having according to an aspect of the present invention,
in dependence upon non-coding or miRNA expression data or an
expression profile (the predictive data or predictive profile)
derived from the population of cells, is not contemporaneous with
the predictive data or predictive profile. Preferably, the cellular
functional effect is separated in time from the predictive data or
predictive profile, by which it is meant from time at which the
predictive data or predictive profile is derived from a population
of cells or sample thereof. The separation in time may be any
suitable time such as at least 15 minutes, at least an hour, at
least 24 hours, at least 1 week or at least 1 month. This
non-contemporaneous nature may be better defined in terms of cell
passages, e.g. separated by at least one passage, at least two
passages, at least 6 passages or at least 12 passages.
[0181] Optionally, according to an embodiment of the present
invention, the cellular functional effect is not demonstrable in
the cell population from which the non-coding RNA or miRNA
expression data or profile derives (at the time a sample is taken
for deriving such expression data or profile), but only after a
passage of time, application of a process or procedure, or of an
intervention on the cell population.
[0182] In a preferred embodiment of the use and method of the
invention, non-coding RNA or miRNA expression data or expression
profile may be derived from a population of cells (e.g. a first
population of cells) at a first occasion for use in identifying,
determining or inferring the propensity for a cellular functional
effect in a population of cells (e.g. a second population of cells)
at a second occasion. The second occasion and first occasion are
typically separated temporally, by process or by intervention on
the cell population. The second population of cells preferably
derives from the first population of cells by the passage of time,
application of a process or an intervention on the first population
of cell.
[0183] It is a preferred embodiment of the invention that the use
and method is for predicting a cellular functional effect (and
optionally a functional outcome) in a population of cells that is
to be subject to a passage of time, application of a process or
procedure or subject to an intervention and in particular to
predict the cellular functional effect (or identify, determine or
infer the propensity thereof) in a population of cells after a
passage of time, application of a process or procedure or after an
intervention, based upon non-coding RNA or miRNA expression data or
expression profiles derived from the population of cells before the
passage of time, the application of the process or procedure or
before the intervention.
[0184] The use according to this embodiment is preferably intended
to enable prediction, determination or inference as to propensity
of a cellular functional effect to occur in a target tissue in
response to treatment by a population of cells leading to a
functional outcome for use in a pre-determined purpose. The target
tissue is preferably tissue of or derived from a patient or
prospective patient and the treating population of cells is
preferably a therapeutic population of cells for cell therapy. The
cellular functional affect according to this general embodiment is
preferably a cellular functional effect as between the target
tissue and the treating population of cells and may be an effect on
the target tissue by the cells or an effect by the treating
population of cells in the environment of the target tissue.
Preferably, this is in order to make a selection as to target
tissue or patients capable of benefiting from administration of the
treating population of cells or cell therapy or in order to make a
determination as to dose of the cell therapy for a particular
patient (e.g. where the extent of the cellular functional effect is
variable and determinable).
[0185] A pre-determined purpose according to this general
embodiment is a purpose or use of the cells that the cellular
functional effect has or is associated with. The pre-determined
purpose may be any purpose to which cells can be put which rely on
some functional effect of the cells (and a functional outcome of
applying the cells). The pre-determined purpose may be, typically,
a cell therapy application.
[0186] A cell therapy application according to this general
embodiment may be, for example, a cancer therapy (such as a
treatment of a cancer with an immune-oncology therapy by
administering therapy with T-cells pre-disposed or modified to
target a tumour or cancer cells, such as chimeric antigen receptor
modified T-cells), regenerative stem cell transplantation (e.g.
bone marrow transplant), regenerative stem cell therapy or
treatment of autoimmune disorders, such as Crohn's disease or
Multiple Scleroses or T-cell mediated immune disorders (e.g. with
stem cells, for example, stem cells pre-disposed or induced with an
immune-modulatory effect) or any other treatment mentioned
hereinbefore.
[0187] The invention according to this general embodiment further
comprises a method of inferring propensity of a cellular functional
effect for a pre-determined purpose as between a treating
population of cells (e.g. a cell therapy) and a target tissue (e.g.
of a patient), the method comprising assaying cells of the target
tissue against a pre-determined miRNA or panel of miRNAs known or
determined to correlate with a desired or intended cellular
functional effect (or extent thereof) of the treating population of
cells on or in the presence of a target tissue, generating miRNA
expression data of the cells for the assayed miRNA or panel of
miRNAs and therefrom identifying, determining or inferring the
propensity for treating the population of cells for the cellular
functional effect on or in relation to that target tissue (or
patient or patient sub-group). This may be achieved by comparing
the expression data generated from the assay with expression levels
(actual, e.g. ranges or thresholds, or relative), trends or
patterns known to be associated (or correlated) with the propensity
for treating the population of cells for the cellular functional
effect on or in relation to that target tissue (or patient or
patient sub-group) and optionally determining from the degree of
similarity or correlation with trends or patterns inferring a
relative or actual propensity. For a panel of miRNAs, the
expression data generated may be compared with a pattern for the
panel and a determination made according to a degree of correlation
with a pattern associated with or correlated with the propensity
for treating the population of cells for the cellular functional
effect on or in relation to that target tissue (or patient or
patient sub-group). Preferably, a selection or decision may be made
to provide an enhanced functional outcome or enhanced efficacy of
the pre-determined purpose based on the determined propensity (or
relative propensity) for the cellular functional effect. That
selection may be, for example, the selection or stratification of
patients suitable for receiving the cell therapy or determination
of dose of cell therapy for a patient or category of patient.
[0188] Optionally, the degree of correlation of the miRNA
expression data may be validated against the functional outcome of
the cellular functional effect in order to attribute an absolute
value or a scale to the miRNA expression data or correlation
thereof with the cellular functional effect or functional outcome
from which a precise prediction or inference can be derived and
thereby a decision or selection may optionally be made.
[0189] The invention further comprises a method of applying a
treating population of cells to a pre-determined purpose in
relation to a target tissue (e.g. of a patient), the method
comprising inferring (or determining) a cellular function effect as
between the treating cells and the target tissue for the
predetermined purpose by the method defined above and in dependence
of the inference (or determination) applying the cells for the
pre-determined purpose.
[0190] The invention according to this general embodiment further
comprises a method of identifying or determining one or more miRNA
the expression of which in a target tissue or cells of or
indicative of the target tissue (e.g. in a prospective patient)
being assayed is correlating with a cellular functional effect of
the cell population to be administered to or for treating the
target tissue (e.g. administered to a patient), preferably for a
predetermined purpose such as cell therapy, for use of said one or
more miRNAs in a panel for identifying, determining or inferring
the propensity of the cellular functional effect in relation to the
target tissue. The method preferably comprises: [0191] identifying
target tissue (or cells from or indicative of target tissue) from
multiple target tissue sources (e.g. different prospective
patients--ideally patients from different patient groups or
patients known or expected to show variable effects) and optionally
sourcing or sampling said target tissue; [0192] Treating and using
a first target tissue sample of each target tissue to isolate total
RNA for miRNA expression profiling (e.g. using Agilent miRNA
microarrays: one version of which, Agilent MiRBase version 16,
contains 1199 human miRNAs; and another version of which, Agilent
MiRBase version 21, contains 2549 human miRNAs) thereby generating
an extensive (e.g. based on at least 100 miRNAs, preferably at
least 800, more preferably at least 1000 and most preferably at
least 2000 miRNAs) miRNA expression profile data set for each
target tissue; [0193] Subjecting each target tissue (e.g. a sample
of the target tissue in vitro or the target tissue in vivo the
latter being subjecting target tissue in each prospective patient)
to a treatment by a treating cell population designed to elicit a
cellular functional effect of the treating cell population in the
target tissue (said treating being, for example, administration to
a patient with a target indication a dose of the treating cell
population) and the extent of the cellular functional effect
monitored by a known or conventional or surrogate means to generate
`response data (e.g. therapeutic effect on patient or patient
target tissue); [0194] Correlating the extensive miRNA expression
profile data set with response data to the treating cell population
by each target tissue (e.g. using correlation methods as are known
in the art); [0195] Identifying correlating miRNA, preferably that
correlate positively or negatively with a correlation coefficient
of at least 7 or at least 8 (e.g. Pearson coefficient or other
accepted correlation measure); and [0196] selecting such
correlating miRNA as candidates for a miRNA expression panel for
the cellular functional effect.
[0197] The invention according to this general embodiment further
comprises a kit for use in identifying, determining inferring a
cellular functional effect as between a treating population of
cells (e.g. for cell therapy) and a target tissue (e.g. of a
prospective patient), the kit comprising primers for use in
quantitative PCR (polymerase chain reaction), primers being for a
miRNA or panel of miRNAs known or identified as correlating with
the cellular functional effect, optionally as further described
above. Optionally, the kit further comprises a protocol and methods
for the PCR assay.
[0198] The invention according to this general embodiment further
provides a companion diagnostic for a cell therapy, the companion
diagnostic comprising a kit, particular to the cell therapy, as
defined above.
[0199] The invention according to this general embodiment further
provides a cell therapy system comprising a cell therapy and a
corresponding companion diagnostic as defined above.
[0200] The present general embodiment further provides use of miRNA
expression data derived from a patient in need of treatment for a
disease to stratify that patient as sufficiently responsive or not
sufficiently responsive to a pre-defined cell therapy for treatment
of the disease. In this case the cellular function could be said to
be the efficacy of the cell therapy in the particular patient.
There is thus further provided use of miRNA expression profiles to
stratify patients according to a particular cell therapy.
[0201] References herein to miRNAs and to particular miRNAs use
miRNA ID codes (miRNA identifiers) following the convention on
naming described on www.mirbase.org, a registry and database of
miRNAs managed by the Griffiths-Jones lab from the University of
Manchester with funding from the BBSRC. The miRNA identifiers are
those valid at 2 May 2016.
EXAMPLES
[0202] Examples are given from different cell types and a variety
of functional outcomes to further illustrate that miRNA expression
can be used to predict downstream functional outcomes and therefore
could be used donor screening, dose determination and cell
therapy.
Example 1--IDO Induction Potential of MSCs
[0203] The induction of indoleamine-2,3-dioxygenase (IDO) has been
shown to correlate with reduced T-cell proliferation and be
responsible for the immunosuppressive action of mesenchymal stem
cells (MSCs) on T-cells. MSCs from different donors (and
potentially different categories of donors) are known to induce IDO
to varying degrees and so the immunosuppressive action of MSCs on
T-cells can vary from donor to donor.
[0204] It was postulated by the inventors that miRNA profile data
may be used to predict or infer IDO induction by MSCs from a
particular donor by screening the donor's cells prior to extensive
cell expansion.
[0205] To identify a panel of miRNA which can form the basis of a
rapid screen for IDO potential, human bone-marrow-derived MSCs from
seven independent donors were sourced from RoosterBio, Inc. The
donor information is summarised in Table 1 below:
TABLE-US-00001 TABLE 1 Donor Age (years) Gender PDL* Cell lot
number 1 22 Male 8.69 00056 2 43 Male 7.32 00009 3 33 Male 7.91
00012 4 29 Female 7.37 00016 5 20 Female 8.86 00071 6 23 Male 8.45
00048 7 25 Male 8.16 0082 *Population doubling levels post
mononuclear cell isolation
[0206] Frozen vials of human bone-marrow-derived MSCs from the
seven independent donors (each containing 1.times.10.sup.7 cells)
were thawed out and re-suspended in 37.degree. C. RoosterBio.TM.
High Performance media (cat. no KT-001) to give a cell suspension
of 1.times.10.sup.6 cells per mL. An aliquot from each donor of
2.times.10.sup.6 cells was generated and immediately centrifuged at
300.times.g for 5 minutes at room temperature. The remaining cells
from the vial were used for seeding tissue culture flasks for cell
expansion (see below). After centrifugation of the 2.times.10.sup.6
cell aliquots, the supernatant was removed by pipette and the cells
re-suspended in 5 mL of ice-cold phosphate buffer saline pH 7.0
(PBS) and centrifuged at 300.times.g for 5 minutes at 4.degree. C.
This step was repeated and the cells re-suspended in 1 mL of
ice-cold PBS, transferred to a 1.5 mL microfuge tube. This was
centrifuged at 300.times.g for 5 minutes at 4.degree. C. and the
supernatant removed by pipette. The resulting cell pellet was
further centrifuged 300.times.g for 1 min at 4.degree. C. to
collect any residual PBS. This was removed by pipette and the cell
pellet snap-frozen in liquid nitrogen and stored at -80.degree. C.
until used for RNA isolation within two weeks of generation.
[0207] All MSC culture was carried out at 37.degree. C. in 95%
standard sea level air: 5% carbon dioxide atmosphere. Human
bone-marrow-derived MSCs from the 7 independent donors were each
seeded in 1.times.T225 tissue culture flasks at 5000 viable cells
per cm.sup.2 and expanded in fully supplemented RoosterBio.TM. High
Performance Media for 3-4 days (80-90% confluence) with no media
change. After expansion in T225 tissue culture flasks, the cells
were harvested by trysinisation using TrypLE.TM. Express 1.times.
enzyme (Thermo Fisher Scientific cat no. 12605036). For the
IDO-induction assay, cells were seeded at confluence at 40,000
cells per cm.sup.2 in 60 mm or 6-well plates (BD Falcon) in 4.4 or
2 mL RoosterBio.TM. High Performance media. After 24 h, media was
changed to RoosterBio.TM. basal media (cat. no. SU-005)
supplemented with 2% Foetal Bovine Serum. After 1 h, cells to be
induced were treated with 10 ng per mL IFN-.gamma. (Thermo Fisher
Scientific cat no. RIFNG50), whilst control cells received no
treatment. After 24 h.+-.1 h, cell culture supernatant was
collected and frozen at 80.degree. C. until assayed for
IDO-induction and activity.
[0208] IDO induction and activity was assayed by quantifying
kynurenine secreted into the cell culture supernatant.
N-formylkynurenine was hydrolysed to kynurenine by 30% (w/v)
trichloroacetic acid. These were then treated with 1% w/v
p-dimethylaminobenzaldehyde in acetic acid, which interacts with
kynurenine to give a yellow product. Kynurenine levels were
assessed by measuring the absorbance in the processed cell culture
supernatants at 480 nM in a spectrophotometer and then comparing
the resulting absorbance units to a 0-10 .mu.g per ml L-kynurenine
standard curve (Sigma-Aldrich cat. no K8625). IDO-induction was
calculated by normalising kynurenine levels to the number of cells
in the well and days of incubation and expressed as pg kynurenine
secreted per cell per day. Levels of IDO-induction are given in
Table 2 below:
TABLE-US-00002 TABLE 2 Donor Control no IFN-.gamma. Plus
IFN-.gamma. 1 0.00 52.2* 2 0.20 67.6 3 0.00 43.6 4 0.20 26.5 5 0.00
14.5 6 0.11 11.8 7 0.00 46.0 *IDO-induction, pg kynurenine secreted
per cell per day
[0209] Total RNA (containing all RNA species, including small
non-coding RNAs such as miRNAs) was isolated from the cell pellets
from cryopreserved cells using the Exiqon miRCURY.TM. RNA Isolation
Kit-Cell & Plant (cat. no. 300110) according to the
manufacturer's instructions v2.2. Total RNA concentrations were
measured using a Nanodrop.TM. 1000 spectrophotometer. RNA purity
and quality were assessed as `pure` based on 260/280 nM and 260/230
nM ratios and `high` RNA Integrity Numbers (RINs) generated using
an Agilent TapeStation 2200--these are summarised in Table 3
below:
TABLE-US-00003 TABLE 3 Donor 260/280 nM ratio.sup.A 260/230 nM
ratio.sup.B RIN.sup.C 1 2.03 2.17 10.0 2 2.09 2.16 9.0 3 2.08 2.24
10.0 4 2.06 2.18 10.0 5 2.06 2.15 10.0 6 2.07 2.00 10.0 7 2.07 2.00
10.0 .sup.A260/280 ratio assesses protein contamination and RNA
with a ratio of ~2.0 is considered `pure` .sup.B260/230 ratio
assesses organic chemical contamination and RNA with a ratio of
2.0-2.2 is considered `pure` .sup.CRINs assess RNA
quality/degradation. RNA with a RIN of .gtoreq.7.0 is considered
good quality, undegraded RNA, RNA with the maximum RIN of 10 being
the highest quality
[0210] For miRNA expression analysis using microarrays, aliquots of
each donor sample RNA were diluted to 50 ng per mL using
nuclease-free water and stored at 80.degree. C. until analysed.
Samples were analysed on the Agilent Technologies, Inc. miRNA
microarray platform (SurePrint G3 Human v16 microRNA 8.times.60K
microarray slides; miRBase v16.0, cat no. G4870A) following the
manufacturer's instructions v1.7. Briefly, one hundred nanograms of
total RNA, from a working solution at 50 ng/.mu.l in nuclease-free
water, were used as input for each microarray experiment. Each
slide contains 8 individual arrays, each array represents 1,349
microRNAs; 1205 human (mapped to 1194 miRNAs miRbase v20) and 144
viral.
[0211] The four key steps of the microarray process were:
[0212] 1. Labelling of RNA with single-colour, Cy3-based
reagent;
[0213] 2. Hybridisation of the labelled RNA samples to the
microarray;
[0214] 3. Wash steps (*NB: the final wash after of the slides in
pre-warmed Wash buffer 2 to 37.degree. C. was carried out with the
outer water bath at 45-50.degree. C., rather than 37.degree. C.);
and
[0215] 4. Slide scanning, data capture and feature extraction
(matching array spots to miRNA IDs) and quality control checks on
the resultant image and data files
[0216] All microarray data passed Agilent quality control metrics
(`good` to `excellent`). Microarray data pre-processing and
normalisation was then carried out with the AgiMicroRNA package in
Bioconductor [details of which are described in Lopez-Romero, P.,
`Pre-processing and differential expression analysis of Agilent
microRNA arrays using the AgiMicroRna Bioconductor library`, BMC
Genomics 12, 64 (2001) and Gentleman, R. C. et al., `Bioconductor:
open software development for computational biology and
bioinformatics`, Genome Biol. 5, R80 (2004)].
[0217] Array quality control was performed using outlier testing
based on the following metrics: [0218] average signal per array
[0219] average background per array [0220] % present (% of miRNAs
where expression is detected on each array) [0221] data
distributions per sample and pairwise
[0222] Normalised miRNA expression levels of detected miRNAs (on
the log 2 scale) were correlated with IDO-induction levels by
correlation analysis using standard R/Bioconductor tools with
Pearson's correlation (`cor.test`) and false discovery multiple
testing correction by the method described in Benjamini, Y. &
Hochberg, `Controlling the False Discovery Rate: A Practical and
Powerful Approach to Multiple Testing`, J R. Stat Soc B 57, 289-300
(1995).
[0223] Across the seven independent donors, the correlation
analysis identified eight miRNAs for which their expression levels
positively or negatively correlated with donor IDO-induction levels
with correlation coefficients .+-.>8.0. These are shown in Table
4 below:
TABLE-US-00004 TABLE 4 miRNA ID Correlation coefficient
hsa-miR-10b-5p +0.89 hsa-miR-136-5p -0.88 hsa-miR-140-3p -0.84
hsa-miR-23a-3p -0.87 hsa-miR-3651 -0.81 hsa-miR-491-3p -0.89
hsa-miR-574-3p -0.93 hsa-miR-574-5p -0.85
[0224] FIGS. 1-8 show correlation plots expression levels vs
IDO-induction for these miRNAs, demonstrating the correlation of
these miRNA across the seven donors for levels of IDO
induction.
[0225] Thus, a candidate group of eight miRNAs, the expression of
which correlate with .+-.>0.8 correlation coefficient values,
with MSC potential for IDO-induction, are identified. Of this
candidate group of eight miRNAs, expression of one is positively
correlated with IDO-induction, whilst the other seven are
negatively correlated with IDO-induction, but in all cases with
correlation coefficient values in excess of 0.8.
[0226] An assay for the screening of donor MSC populations to infer
(or determine) IDO-induction potential (or relative induction
potential as between MSCs of more than one donor) can be carried
out using one or a panel of two or more of the identified eight
candidate miRNAs and preferably up to six of the candidate miRNAs.
These miRNAs could be selected and used to screen donor MSCs to
predict and pre-select suitable donor cells with the highest
immunosuppressive potential for further development as cell
therapies.
Example 2--Migration Propensity of CD34.sup.+ Cells
[0227] miRNA expression profiles from isolated human CD34.sup.+
cells from eight independent donors with different migration
potentials were generated. These cell were then assayed in a
SDF-1-based migration assay using standard methodologies and the
percent migration determined. Correlation analysis was then carried
out to identify miRNAs for which their expression correlated with
and predicted the ability of the CD34.sup.+ cells to migrate
towards a chemoattractant gradient.
[0228] Two candidate miRNAs were identified, the expression of
which positively correlated with percent migration with a
correlation coefficient .+-.>0.8. These are shown in Table 5
below:
TABLE-US-00005 TABLE 5 miRNA ID Correlation coefficient
hsa-miR-1471 +0.85 hsa-miR-1288-3p +0.93
[0229] FIGS. 9 and 10 show correlation plots expression levels vs
percent migration for these miRNAs.
[0230] Thus, the above-mentioned miRNAs can be used as basis for
screening CD34+ cells or blood/bone marrow cell samples for
propensity to migrate whereby the amount of cell material for
expansion, the extent of expansion and/or the dose of cells for
administration in autologous cell therapy.
[0231] The invention has been described with reference to a
preferred embodiment. However, it will be appreciated that
variations and modifications can be effected by a person of
ordinary skill in the art without departing from the scope of the
invention.
* * * * *
References