GB2605113A - Functional prediction - Google Patents

Functional prediction Download PDF

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GB2605113A
GB2605113A GB2209255.5A GB202209255A GB2605113A GB 2605113 A GB2605113 A GB 2605113A GB 202209255 A GB202209255 A GB 202209255A GB 2605113 A GB2605113 A GB 2605113A
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mirna
cells
functional effect
cell
cellular functional
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GB202209255D0 (en
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J Mallinson David
R Dunbar Donald
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Sistemic Scotland Ltd
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Sistemic Scotland Ltd
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Priority claimed from GB1608081.4A external-priority patent/GB2550136A/en
Publication of GB202209255D0 publication Critical patent/GB202209255D0/en
Publication of GB2605113A publication Critical patent/GB2605113A/en
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Abstract

A method of using non-coding RNA (e.g. miRNA) expression data or expression profiles to identify, determine or infer propensity for a cellular functional effect for a pre-determined purpose; the method comprising assaying against pre-determined non-coding RNAs known to correlate with the cellular functional effect, generating non-coding RNA expression data for the assayed non-coding RNA panel and from the non-coding RNA expression data identifying, determining or inferring a propensity for the cellular functional effect. Also claimed is a method for screening cell populations of donor-derived cells (e.g. stem cells, MSCs) for use in treatment of a condition comprising inferring a cellular function effect pertinent to the treatment and selecting cells for use in the treatment; a kit comprising primers for quantitative PCR, the primers being for a non-coding RNA. Examples are provided correlating miRNA expression data with the induction of IDO by IFN-γ in MSCs and migration propensity of CD34+ cells towards SDF-1.

Description

Functional Prediction
FIELD OF THE INVENTION
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
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.
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.
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.
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-20121025709.
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
There is a need for improvements in the predictability of functional outcome in cell applications in biological systems and for making selection decisions.
It is an object of this invention to provide a method for predicting the fimctional outcome the application of cells or interventions on cells in a biological system.
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.
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
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.
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 predetermined 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.
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.
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.
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.
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.
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.
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 con-elates with the cellular fimctional effect to a degree that corresponds with one of a plurality of possible doses or dosage regimen.
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.
In a tenth aspect of the invention, there is provided a method of identifying or determining one or more non-coding RNA (or m i RNA) 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: Sourcing cell populations intended for effecting the cellular functional effect from multiple sources (e.g. multiple donors or multiple production batches of different provenance); 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)1; 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': Correlating the extensive non-coding (or miRNA) expression profile data set with response data for each cell population; 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 selecting such correlating non-coding RNA (or miRNA) as candidates for a miRNA expression panel for the cellular functional effect.
ADVANTAGES OF THE INVENTION
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
The invention concerns the use of non-coding expression data or expression profiles to identify, deteimine 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 predetermined 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.
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 (siaRNA), 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.
The term miRNA (rnicroRNA) may include miRNA molecules and either or both miRNA precursors and mature miRNAs as is apparent from the context, but are preferably mature miRNAs.
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).
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 m1 RNA and preferably up to six miRNA.
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.
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.
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.
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.
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 1MSCs, 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), 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.
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.
The invention further comprises a method of applying a cell population to a pre-detenn ined 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. 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.
The invention further comprises the use of a panel of miRNA (or miRNA adapted for assay form) in an assay for inferring a cellular fiinctional effect for which panel of miRNA the expression has been determined as correlating with a cellular functional effect.
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 me-defined mid 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.
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.
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 myelorna cells. Chinese banister 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.
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.
The present invention in its broadest sense comprises at least two general embodiments.
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.
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.
By expression data, it is meant expression levels relative expression levels or expression profiles or patterns of miRNA of the cells.
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).
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.
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).
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 intcrventionally from the cells or cell population from which the inference or identification is being made (by means of assaying for miRNA expression).
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 I,000 Daltons or less than 500 Daltons, or non-polymeric, or a biological entity (e.g. a biological macromolecule, such as a lipid, an oligonueleofide, 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 meehanochemical 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. -10-
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 predetermined 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).
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 predetermined 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 predetermined purpose may be, for example, a bioprocess application, a regenerative medicine application, cell therapy, cell growth or donor selection.
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 predetermined 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 predetermined 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-detained purpose.
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.
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: Sourcing cell populations intended for effecting the cellular functional effect from multiple sources (e.g. multiple donors or multiple production batches of different provenance); Treating and using a first sample of each cell population to isolate total RNA for miANA expression profiling (e.g. using Agilent miRNA microarrays: one version of which, Agilent NERBase 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, 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); Correlating the extensive miRNA expression profile data set with response data for each cell population (e.g. using correlation methods as arc known in the art); -12-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 selecting such correlating miRNA as candidates for a miRNA expression panel for the cellular functional effect.
The invention further comprises a method of applying cells to a predetermined purpose, the method comprising identifying, determining or inferring a cellular function effect of the cells for the predetennined purpose by the method defined above and in dependence of the identification, detennination or inference applying the cells for the pre-determined purpose.
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.
The invention according to this embodiment further comprises a cell population selected or adapted to have a propensity or pre-detennined 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.
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 -13-enzymes, for example the induction by interferon gamma of indoleamine-2,3-dioxygenase (IDO) in mesenchymal stem cells.
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.
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 arc 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.
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, eliondrocytes, 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 -14-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.
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). ehondrocytes (chondrogenesis) and osteocytes (osteogenesis). A kit may typically be for use in quantitative PCR (polyrnerase 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). ehondrocytes (chondrogenesis) and osteocytes (osteogenesis). The kit may further comprise protocol and methods for the PCR assay. The kit may farther 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 predefined or desired cell lineage such as one of adipocytes (adipogenesis), ehondrocytes (chondrogenesis) and osteocytes (osteogenesis) and/or ranges of expression levels corresponding with different extents of differentiation into one of adipocytes (adipogenesis), ehondroeytes (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.
In one embodiment, the pre-determined purpose is a bioprocess application.
A b oprocess 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.
-15 -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 immmm-oncology therapy. The therapy may be heterologous, or homologous and may be allogenic or autologous.
in one embodiment, the pre-determined purpose is cell therapy, for example stem cell therapy, for example regenerative stem cell thcrapy. 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.
The cell therapy may optionally be for autologous, homologous or heterologous therapy, preferably homologous (e.g. autologous or allogenic).
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 bioproccss application or a therapeutic effect.
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.
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 nURNA 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 -16-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.
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.
Preferably the cells are human cells. Preferably the cells are stem cells or T-cells.
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.
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.
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 ti SRIC 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 predisposed or induced with an immuno-modulatory 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 inurnam- -17-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.
In one embodiment, the use and method is directed toward T-cells, autologous or allogen lc, optionally modified, for use in therapy.
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.
In one embodiment, the use and method is directed toward mesenchymal stem cells for use in therapy.
In one embodiment, the use and method is directed toward haematopoictie stem cells. Such cells may be derived from bone marrow or peripheral blood. Such cells may comprise cells that are CD34+ cells.
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.
There are now described more particular embodiments of the invention falling within this general embodiment.
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-7). IDO induction is considered a surrogate for the inumme-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.
-18 -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.
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 indoleaminc-2,3-dioxygenasc (IDO) induction by interferon-gamma (IFN-y) (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-y, 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 1FN-y (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-y (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 pan& 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-y (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 1D0 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 predetermined threshold of propensity for IDO induction by IFN-y 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 -19-better demonstrate propensity for MO induction by IFN-y (that is, have a better relative propensity) for use in therapy.
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.
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 1DO induction by 1FN-y in the MSCs by the method defined above and in dependence of the inference (or determination) applying the MSCs for the therapy.
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 MO 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 1DO induction.
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.
The propensity may be a relative propensity or an absolute propensity. Preferably, the MSCs having a propensity for 1DO 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 1DO 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-y induction of MO 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).
By expression data it is meant expression levels, relative expression levels or expression profiles or patterns of miRNA of the cells.
-20 -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-y. More preferably, the miRNA or panel of miRNAs have expression that correlates with MO induction by IFN-y with a correlation coefficient equal to or greater than 7.0, more preferably equal to or greater than 8.0 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-y (e.g. hsa-miR-10b-5p) and/or at least one miRNA negatively correlated with IDO induction by IFN-y (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.
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 (1FN-y) 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 MO induction. The kit may further comprise protocol and methods for the PCR assay. The kit may further comprise a set of miRNA samples from A4SCs, 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 mi RNA arc preferably as defined above.
Alternatively, the cells may be instead of T cells, B cells, NK cells or DC cells, which offer a potential therapy autoimmtme disorders (including GVHD, but also Multiple Scleroses or Crohn's disease).
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 -21 -propensity or relative propensity of those MSCs for differentiation into adipocytes (adipogenesis), chondrocytes (chondrogcncsis) 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.
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.
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 chondrocvtes) 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.
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 m iRNAs and identifying, determining or inferring therefrom the propensity of the MSCs for differentiation into one of ad ipocytes (adipogenesis), chondrocytes (chondrogenesis) ancUor 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 -22 -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.
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.
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).
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 -23 -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).
Preferably the method and MSCs are for the treatment of conditions treatable by regenerative cell therapy (especially regenerative stern 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 pit-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 ostcoregenerative therapy, such as in the treatment of fractures or osteoporosis.
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 osteocvres (osteogenesis), which said expression profile meets predetermined 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).
By expression data, it is meant expression levels, relative expression levels or expression profiles or patterns of miRNA of the cells.
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.
-24 -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.
Preferably, the miRNA expression data and miRNA or panel of miRNAs for use in identifying, determining or inferring the propensity or relative propensity of NISCs to differentiate into osteocytes (osteogenesis) preferably includes at least one miRNA positively correlated with osteogenesis and/or at least one miRNA negatively con-elated with osteogenesis. Preferably, the panel of miRNA comprises at least two miRNA and more preferably up to six miRNAs.
Further, according to this particular embodiment, there is provided a kit for use to identify, determine or infer the propensity or relative propensity of NISCs 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 ostcocytes (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 arc preferably as defined above for each purpose.
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).
-25 -CD34+ cell populations are typically derived from blood or bone marrow and arc 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 hacmatopoictic 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.
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 engraftmcnt potential and dosing. However, the assay is convoluted and time consuming and incompatible with the time pressures of autologous CD34+ cell therapy.
This particular embodiment is directed toward a rapid screen that is a surrogate for an in viiro 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-I according to an in vitro SDF-I migration assay. The use is directed toward the engraftment of stem cells in autologous stem cell therapy.
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 ischcmia (e.g. to increase exercise capacity), myocardial infarction and ischcmic stroke, multiple sclerosis and autoimmune disorders. The application of this particular embodiment has particular utility in autologous therapy.
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.
Optionally, according to this particular embodiment, the miRNA expression profile may be validated against the SDF-I 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.
-26 -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-I 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.
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.
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-I 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.
-27 -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.
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.
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-I 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-I 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).
By expression data it is meant expression levels, relative expression levels or expression profiles or patterns of miRNA of the cells.
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.
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.
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, -28 -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-I 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-I 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.
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.
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 arc 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 Unmunotherapies, especially immuno oncology therapies, such as cell-based adoptive transfer of T-cells.
T-cell populations (or modified T-cell populations) for use in such therapeutic applications require to be expanded to provide therapeutic doses for patients.
Indications for autologous or allogenic T-cell based immunotherapies, such as by cell-based adoptive transfer of T-cells, include autoim mune disorders and cancers such as solid tumors, cervical cancer, lymphoma, leukamias, bile duct cancer, neuroblastoma, lung cancer, breast cancer, sarcoma, melanoma. CD19-expressing hacmatologic 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 -29 -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 deterrriining the quantity of donor T-cell material required to expand to a therapeutic dose in a pre-determined time.
Optionally, according to this particular embodiment, the miRNA expression profile may be validated against proliferation rate whereby die miRNA expression profile levels may represent indicators of absolute proliferation potential and thus a decision or selection may be made accordingly.
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. 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 -30 - (or determination), modifying and expanding the T-cells to a therapeutic dose and applying the T-cell dose for the therapy.
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.
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 CART-cell therapy.
The propensity may be a relative propensity or an absolute propensity. Preferably, the T-cell populations haying 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).
By expression data, it is meant expression levels, relative expression levels or expression profiles or patterns of miRNA of the cells.
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.
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.
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 -31 -proliferation potential or for other consequential or corresponding purpose. A kit may typically bc for usc in quantitative PCR (polymerasc 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.
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.
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).
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.
Thus, the miRNA expression data can be used to stratify a patient population for receiving the cell therapy.
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 -32 -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.
By expression data, it is meant expression levels, relative expression levels or expression profiles or patterns of miRNA of the cells.
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).
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.
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).
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).
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 -33 -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).
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 predetermined purpose may be, typically, a cell therapy application.
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 1-cells pre-disposed or modified to target a tumour or cancer cells, such as chimeric antigen receptor modified 1-cells), regenerative stem cell transplantation (e.g, bone marrow transplant), regenerative stem cell therapy or treatment of autoinunune disorders, such as Crohn's disease or Multiple Scleroses or 1-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.
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 detemfined 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 -34 -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.
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.
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.
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: 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; 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 microan-ays: 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, -35 -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; 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); 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); - 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 - selecting such correlating miRNA as candidates for a m iRNA expression panel for the cellular functional effect.
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 thriller comprises a protocol and methods for the PCR assay.
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.
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 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 -36 -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.
References herein to miRNAs and to particular miRNAs use miRNA ID codes (miRNA identifiers) following the convention on naming described on www.mirbasc.ore. 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'd May 2016.
EXAMPLES
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 15 therapy.
Example I -IDO induction potential of MSCs The induction of indolcaminc-2,3-dioxygcnasc (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 TDO to varying degrees and so the immunosuppressive action of MSCs on T-cells can vary from donor to donor.
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.
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 I below: Table 1 Donor Age (years) Gender PDL* Cell lot number I 22 Male 8.69 00056 2 43 Male 7.32 00009 3 33 Male 7.91 00012 4 29 Female 7.37 00016 20 Female 8.86 00071 6 23 Male 8.45 00048 -37 - 7 25 Male 8.16 0082 *Population doubling levels post mononuclear cell isolation Frozen vials of human bone-marrow-derived MSCs from the seven independent donors (each containing 1 x 107 cells) were thawed out and re-suspended in 37°C RoosterBirnm High Performance media (cat. no KT-001) to give a cell suspension of 1 x 106 cells per mL. An aliquot from each donor of 2 x 10' cells was generated and immediately centrifuged at 300rng 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 x 106 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 300xg for 5 minutes at 4°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 300xg for 5 minutes at 4°C and the supernatant removed by pipette. The resulting cell pellet was further centrifuged 300xg for lmin at 4°C to collect any residual PBS. This was removed by pipette and the cell pellet snap-frozen in liquid nitrogen and stored at -80°C until used for RNA isolation within two weeks of generation.
All MSC culture was carried out at 37°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 I x T225 tissue culture flasks at 5000 viable cells per cm2and expanded in fully supplemented RoosterBioni High Performance Media for 3-4 days (80-90% confluence) with no media change. After expansion in 1225 tissue culture flasks, the cells were harvested by trysinisation using TrypLETNI Express 1 x enzyme (Thermo Fisher Scientific cat no. 12605036). For the IDO-induction assay, cells were seeded at confluence at 40,000 cells per cm' in 60 mm or 6-well plates (BD Falcon) in 4.4 or 2 mL RoosterBio' High Performance media. After 24h, media was changed to RoosterBiol" basal media (cat.
no. SU-005) supplemented with 2% Foetal Bovine Serum, After 111, cells to be induced were treated with 10 ng per mL IFN-y (Thermo Fisher Scientific cat no. RIFNG50), whilst control cells received no treatment. After 2411±1h, cell culture supernatant was collected and frozen at 80°C until assayed for IDO-induction and activity.
MO induction and activity was assayed by quantifying kynurenine secreted into the cell culture supernatant. N-fonnylkynurenine was hydrolysed to kynurenine by 30% (w/v) trichloroacetic acid. These were then treated with 1% w/v pdimethylaminobenzaldehyde in acetic acid, which interacts with kynurcnine 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 -38 -absorbance units to a 0-10 1..ig per ml L-kynurenine standard curve (Sigma-Aldrich cat. no 1(8625). 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 MO-induction are given in Table 2 below:
Table 2
Donor Control no IFN-y Plus IFN-y 1 0.00 52.2* 2 0.20 67.6 3 0.00 43.6 4 0.20 26.5 0.00 14.5 6 0.11 11.8 7 0.00 46.0 *1W-induction. pg kmurenine secreted per cell per day 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 miRCURYtm RNA Isolation Kit-Cell 8z. Plant (cat. no. 300110) according to the manufacturer's instructions v2.2. Total RNA concentrations were measured using a Nanodroptm 1000 spectrophotometer. RNA purity and quality were assessed as 'pure' based on 260/280nM and 260/230nM ratios and 'high' RNA Integrity Numbers (RINs) generated using an Agilent TapeStation 2200 -these are summarised in Table 3 below:
Table 3
Donor 260/280nM ratio' 260/230nM ratio' AIN(' 1 2.03 2.17 10.0 2 2.09 2.16 9.0 y 2.08 2.24 10.0 4 2.06 2.18 10.0 2.06 2.15 10.0 -39 - 6 2.07 2.00 10.0 7 2.07 2.00 10.0 A -260/280 ratio assesses protein coma ation and RNA ha ra to of -2.0 is considered 'pure' B -260/230 ratio assesses organic chemical contamination and RATA with a ratio of 2.0-2.2 is considered 'pure' C -RINs assess RNA quality/degradation. RNA with a RIN of >7.0 is considered good quality, undegraded RNA, RNA with the maximum RINqf 10 being the highest quality 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°C until analysed. Samples were analysed on the Agilent Technologies, Inc. miRNA microarray platform (SurePrint G3 Human v16 microRNA 8x601C 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/u1 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.
The four key steps of the microarray process were: 1. Labelling of RNA with single-colour, Cy3-based reagent; 2. Hybridisation of the labelled RNA samples to the microarray, 3. Wash steps (*NB: the final wash after of the slides in pre-warmed Wash buffer 2 to 37°C was carried out with the outer water bath at 45-50°C, rather than 37°C), and 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 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', BVIC Genomics 12, 64 (2001) and Gentleman, R. C. et at, 'Bioconductor: open software development for computational biology and bioinfonnatics', Genome Biol. 5, R80 (2004)].
Array quality control was performed using outlier testing based on the following metrics: * average signal per array
* average background per array
* l<) present (% of miRNAs where expression is detected on each array) -40 - * data distributions per sample and painvise Normalised miRNA expression levels of detected miRNAs (on the log2 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 Sac B 57, 289-300 (1995).
Across the seven independent donors, the correlation analysis identified eight miRNAs for which their expression levels positively or negatively correlated with donor MO-induction levels with correlation coefficients +>8.0. These are shown in Table 4 below:
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-nUR-365 I -0.81 h sa-rn iR-491-3p -0.89 hsa-miR-574-3p -0.93 hsa-miR-574-5p -0.85 Figures 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 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 IDOinduction, but in all cases with correlation coefficient values in excess of 0.8.
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 -41 -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-mi2ration propensity of CD34+ cells miRNA expression profiles from isolated human CD34+ 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 mid 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+ cells to migrate towards a chemoattractant gradient.
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
miRNA ID Correlation coefficient hsa-miR-1471 +0.85 hsa-miR-1288-3p +0.93 Figures 9 and 10 show correlation plots expression levels vs percent migration for these miRNAs.
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.
Further aspects and/or embodiments of the invention are described in the following clauses: Clause 1. Use of miRNA expression data or expression profiles to identify, determine or nfer propensity for a cellular functional effect for a pre-determined purpose.
Clause 2. A use according to clause 1, which is the use of non-coding RNA expression data of a cell population to identify, determine or infer the propensity of a cellular functional effect of that cell population.
-42 -Clause 3. A use according to clause I or clause 2, wherein the non-coding RNA expression data is of a prc-deteimined non-coding RNA or panel of non-coding RNA the expression of which is known to correlate with the cellular functional effect or a known surrogate or assay thereof Clause 4. A use according to clause 3, which can be effected by assaying against the pre-5 determined non-coding RNA or panel of non-coding RNA Clause 5. A use according to clause 3, wherein the panel of non-coding RNA comprises at least two non-coding RNA and preferably up to six non-coding RNA.
Clause 6. A use according to clause 5, 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.
Clause 7. A use according to any one of the preceding clauses where].n the pre-determined purpose is selected from a bioprocess applications, a cell therapy application, patient stratification for cell therapy, cell growth and donor selection.
Clause 8. A use according to any one of the preceding clauses, 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 Clause 9. A use according to any one of the preceding clauses which is further for making a selection or treatment decision based upon the identification, determination or inference of propensity for the cellular functional effect.
Clause ID, A method of inferring propensity for a cellular functional effect for a pre-determined purpose, the method comprising assaying 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.
Clause 11. A method according to clause 10, 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.
-43 -Clause 12. A method according to clause 10 or clause 11, which is for inferring a cellular fiinctional effect in a cell population by assaying that cell population against the predetermined non-coding RNA or panel of non-coding RNAs.
Clause 13. A method according to any one of clauses 10 to 12, wherein the panel of non-coding RNA comprises at least two non-coding RNA and preferably up to six non-coding RNA.
Clause 14. A method according to clause 13, wherein at least one non-coding RNA is positively correlated and at least one non-coding RNA is negatively correlated with the cellular ftinctional effect.
Clause 15. A method according to any one of clauses 10 to 14, 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.
Clause 16. A method according to any one of clauses 10 to 15, 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, engrafiment potential, immunosuppress ve activity, migration potential, response to activity inducing agents or response to assays.
Clause 17. A method according to any one of clauses 10 to 16, which further comprises making a selection or treatment decision based upon the identification, determination or inference of propensity for the cellular functional effect.
Clause 18. A use or method according to any one of the preceding clauses, which is for inferring a cellular functional effect in a cell population using the non-coding RNA expression data from that cell population for a predetermined non-coding RNA or panel of non-coding ANAs, wherein the cell population are human cells.
Clause 19. A use or method according to clause 18, wherein the cells are stem cells or T-cells.
Clause 20. A use or method according to clause 19 therein the cells are T-cells which are optionally modified for surface antigenic activity.
Clause 21. A use or method according to clause 20, wherein the cellular functional effect comprises migratory potential of the T-cells to target tumour cells and the pre-determined purpose is the treatment of a cancer.
Clause 22. A use or method according to clause 19, wherein the cells are stem cells.
-44 -Clause 23. A use or method according to clause 22, which cells are stern cells selected from MSCs, induced pluripotcnt stem cells or hacmatopoictic stem cells such as CD34+ cells.
Clause 24. A use or method according to clause 23, wherein the cells are MSCs.
Clause 25. A use or method according to clause 23 or clause 24, wherein the cellular functional effect is differentiation into chondrocytes and the pre-determined purpose is regenerative treatment of cartilage tissue.
Clause 26. A use or method according to clause 23 or clause 24, wherein the cellular functional effect is differentiation into ostcocytes and the pre-determined purpose is regenerative treatment of bone tissue.
Clause 27. A use or method according to clause 23 or clause 24, wherein the cellular fiinctional effect is secretion of paracrine factors and the pre-determined purpose is regenerative treatment of target tissue.
Clause 28. A use or method according to clause 27, wherein the cellular functional effect is secretion of one or more paracrine factors known to influence a pre-determined target tissue regeneration and the pre-determined purpose is regenerative treatment of target tissue.
Clause 29. A use or method according to clause 28, wherein the paracrine factors are growth factors for one of vascular smooth muscle and/or cardiomyocytes and the pre-determined purpose is regenerative treatment of cardiac or vascular tissue.
Clause 30. A use or method according to clause 24, wherein the cellular functional effect is the TOO induction of the MSCs by interferon-gamma and the pre-determined purpose is treatment of T-cell proliferation-mediated indications or Graft vs Host Disease.
Clause 31. A use or method according to clause 23, 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.
Clause 32. A use or method according to clause 31, wherein the stem cells are CD34+ cells.
Clause 33. A use or method according to clause 18, wherein the pre-determined purpose is for the treatment of one of an immune disorder such as Crolm's disease, rheumatoid arthritis, lupus, TBD or MS, a cancer, a cardiovascular disease or a tissue-degenerative disorder.
-45 -Clause 34. A method of applying a cell population to a pre-determined purpose, the method comprising inferring a cellular function effect for the predetermined purposc by the method of any one of clauses 10 to 33 and applying the cell population for the pre-determined purpose.
Clause 35. 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.
Clause 36. A method for determining the dose of a population of cells (e.g stem cells, such as TVISCs or T-cells) for administration to a patient for treating a condition which depends upon a cellular functional effect, the method comprising measuring an non-coding RNA expression profile for the cells 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 to the cellular functional effect an actual dose to be administered to a patient.
Clause 37. 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.
Clause 38. A method according to clause 36 or clause 37, wherein the treatment is one of autologous cell therapy or allogenic cell therapy.
Clause 39. A population of cells selected to have a non-coding RNA expression profile for a pre-defined non-coding RNA or panel of non-coding RNAs that correlates with a pre-defined cellular functional effect optionally for a pre-determined purpose.
Clause 40. A population of cells according to clause 39, derived from a donor of said cells, the donor having been selected based upon a non-coding RNA expression profile for a pre-defined non-coding RNA or panel of non-coding RNAs that correlates with a pre-defined cellular functional effect.
-46 -Clause 41. A cell population according to clause 39 or clause 40 for use in therapy or diagnosis of the human or animal body.
Clause 42. 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.
Clause 43. A cell population according to any one of clauses which are human cells.
Clause 44. A cell population according to clause 42 or clause 43 which cells are stem cells or T-cells.
Clause 45. A cell population according to clause 44, wherein the cells are T-cells which are optionally modified for surface antigenic activity.
Clause 46. A cell population according to clause 44, for the treatment of a cancer.
Clause 47. A cell population according to clause 44, wherein the cells are stem cells.
Clause 48. A cell population according to clause 44, which cells arc stem cells selected from MSCs, induced pluripotent stem cells or haematopoietic stem cells such as CD34+ cells.
Clause 49. A cell population according to clause 48, wherein the stem cells are MSCs.
Clause 50. A cell population according to any one of clauses 47 to 49, wherein the cellular functional effect is differentiation into chondrocytes and the pre-detennined purpose is regenerative treatment of cartilage tissue.
Clause 51. A cell population according to any one of clauses 47 to 49, wherein the cellular functional effect is differentiation into osteocytes and the pre-determined purpose is regenerative treatment of bone tissue.
Clause 52. A cell population according to any one of clauses 47 to 49, wherein the cellular functional effect is secretion of paracrine factors and the pre-determined purpose is regenerative treatment of target tissue.
-47 -Clause 53. A cell population according to clause 52, wherein the cellular functional effect is secretion of one or more paracrinc factors known to influence a pre-determined target tissue regeneration and the pre-determined purpose is regenerative treatment of target tissue.
Clause 54. A cell population according to clause 52, wherein the paracrine factors are growth factors for one of vascular smooth muscle and/or cardiomyocytes and the pre-determined purpose is regenerative treatment of cardiac or vascular tissue.
Clause 55. A cell population according to clause 49, wherein the cellular functional effect is the IDO induction of the MSCs by interferon-gamma and the pre-determined purpose is treatment of T-cell proliferation-mediated indications or Graft vs Host Disease.
Clause 56. A cell population according to clause 47 or clause 48, wherein the cellular functional effect is migration toward stromal cell-derived-factor 1 (SDF-1) and the predetermined purpose is selection of donors of stem cells for engraftment potential Clause 57. A cell population according to clause 56 wherein the stem cells are CD34-F cells.
Clause 58. A cell population according to any one of clauses 39 to 42, which is for the treatment of one of an immune disorder such as Crohn's disease, rheumatoid arthritis, lupus, TBD or MS, a cancer, a cardiovascular disease or a tissue-degenerative disorder.
Clause 59. 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 panel of non-coding RNAs known or identified as correlating with the cellular functional effect.
Clause 60. A kit according to clause 59, which further comprises a protocol and methods for the PCR assay.
Clause 61. A method of identifying or determining one or more non-coding RNA 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 in a panel for identifying, determining or inferring the cellular functional effect, the method comprising: -48 - - Sourcing cell populations intended for effecting the cellular functional effect from multiple sources (e.g. multiple donors or multiple production batches of different provenance); - Treating and using a first sample of each cell population to isolate total RNA for non-coding RNA expression profiling thereby generating an extensive (e.g. based on at least miRNAs, preferably at least 800, more preferably at least 1000 and most preferably at least 2000 miRNAs) non-coding RNA expression profile data set for each cell population; - 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', - Correlating the extensive non-coding RNA expression profile data set with response data for each cell population; - Identifying correlating non-coding RNA, preferably that correlate positively or negatively with a correlation coefficient of at least 7 or at least 8; and selecting such con-elating non-coding RNA as candidates for a non-coding RNA expression panel for the cellular functional effect.
Clause 62. A use, methods, population of cells or kit according to any onc of the preceding clauses, wherein the non-coding RNA are miRNA 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.
-49 -

Claims (25)

  1. CLAIMS: I. Use of miRNA expression data or expression profiles to identify, determine or infer propensity for a cellular functional effect for a pre-determined purpose.
  2. 2. A use as claimed in claim 1, which is the use of miRNA expression data of a cell population to identify, determine or infer the propensity of a cellular functional effect of that cell population.
  3. 3 A use as claimed in claim 1 or claim 2, wherein the miRNA expression data is of a pre-determined miRNA or panel of miRNA the expression of which is known to correlate with the cellular functional effect or a known surrogate or assay thereof
  4. 4. A use as claimed in claim 3, which can be effected by assaying against the pre-determined miRNA or panel of miRNA, wherein the panel of miRNA preferably comprises at least two miRNA and preferably up to six miRNA.
  5. 5. A use as claimed in claim 4, wherein at least one miRNA is positively correlated and at least one miRNA is negatively correlated with the cellular functional effect.
  6. 6. A use as claimed in any one of the preceding claims 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. 7. A use as claimed in any one of the preceding claims, 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. 8. A use as claimed in any one of the preceding claims which is further for making a selection or treatment decision based upon the identification, determination or inference of propensity for the cellular functional effect.-50 -
  9. 9. A method of inferring propensity for a cellular functional effect for a predetermined purpose, the method comprising assaying 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 from the miRNA expression data identifying, determining or inferring a propensity for the cellular functional effect.
  10. 10. A method as claimed in claim 9, wherein the identifying, deteimining 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.
  11. 11. A method as claimed in claim 9 or claim 10, which is for inferring a cellular functional effect in a cell population by assaying that cell population against the predetermined miRNA or panel of miRNAs, wherein the panel of miRNA preferably comprises at least two miRNA and preferably up to six miRNAs.
  12. 12 A method as claimed in claim I I, wherein at least one miRNA is positively correlated and at least one miRNA is negatively correlated with the cellular functional 20 effect.
  13. 13. A method as claimed in any one of claims 9 to 12, 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
  14. 14. A method as claimed in any one of claims 9 to 13, wherein the cellular fimctional 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, inununosuppressive activity, migration potential, response to activity inducing agents or response to assays.
  15. IS. A method as claimed in any one of claims 9 to 14, which further comprises making a selection or treatment decision based upon the identification, determination or inference of propensity for the cellular functional effect. -51 -
  16. 16. A use or method as claimed in any one of the preceding claims, which is for inferring a cellular functional effect in a cell population using the miRNA expression data from that cell population for a predetermined miRNA or panel of miRNAs, wherein the cell population are human cells, preferably stcm cells or T-cells.
  17. 17. A use or method as claimed in claim 16, wherein the cells are T-cells which are optionally modified for surface antigenic activity and wherein the cellular functional effect preferably comprises migratory potential of the T-cells to target tumour cells and the pre-determined purpose is the treatment of a cancer.
  18. 18. A use or method as claimed in claim 16, wherein the cells are stem cells, preferably selected from MSCs, induced pluripotent stem cells or haematopoietic stem cells such as CD34+ cells.
  19. 19. A use or method as claimed in claim 18, wherein the cells are MSCs and the cellular functional effect is optionally differentiation into chondrocytcs and the predetermined purpose is optionally regenerative treatment of cartilage tissue, or the cellular functional effect is optionally differentiation into osteocytes arid the pre-determined purpose is optionally regenerative treatment of bone tissue.
  20. 20. A use or method as claimed in claim 16, wherein the pre-determined purpose is for the treatment of one of an immune disorder such as Crolm's disease, rheumatoid arthritis, lupus, MD or MS, a cancer, a cardiovascular disease or a tissue-degenerative disorder.
  21. 21. A method of applying a cell population to a pre-detennined purpose, the method comprising inferring a cellular function effect for the predetermined purpose by the method of any one of claims 9 to 20 and applying the cell population for the predetermined purpose.
  22. 22. 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 thither 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 -52 -donors for further donation of cells) for use in the treatment of the indication or the further manipulation.
  23. 23. 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 miRNA expression profile for the cells for a pre-determined miRNA or panel of 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 to a predetermined dose for a pre-determined propensity to the cellular functional effect an actual dose to be administered to a patient.
  24. 24. A population of cells selected to have a miRNA expression profile for a predefined miRNA or panel of miRNAs that correlates with a pre-defined cellular functional effect optionally for a pre-determined purpose, preferably for use in therapy or diagnosis of the human or animal body.
  25. 25. 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 comprising: - Sourcing cell populations intended for effecting the cellular functional effect from multiple sources (e.g. multiple donors or multiple production batches of different provenance); - Treating and using a first sample of each cell population to isolate total RNA for miRNA expression profiling 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; - 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': -5.3 -Correlating the extensive miRNA expression profile data set with response data for each cell population; Identifying correlating miRNA, preferably that correlate positively or negatively with a correlation coefficient of at least 7 or at least 8; and selecting such correlating miRNA as candidates for a miRNA expression panel for the cellular functional effect -54 -
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